Osmoregulation and Excretion
Erik Hviid Larsen,*1 Lewis E. Deaton,2 Horst Onken,3 Michael O’Donnell,4 Martin Grosell,5
William H. Dantzler,6 and Dirk Weihrauch7
ABSTRACT
The article discusses advances in osmoregulation and excretion with emphasis on how multicellular animals in different osmotic environments regulate their milieu intérieur. Mechanisms of energy
transformations in animal osmoregulation are dealt with in biophysical terms with respect to water and ion exchange across biological membranes and coupling of ion and water fluxes across
epithelia. The discussion of functions is based on a comparative approach analyzing mechanisms
that have evolved in different taxonomic groups at biochemical, cellular and tissue levels and
their integration in maintaining whole body water and ion homeostasis. The focus is on recent
studies of adaptations and newly discovered mechanisms of acclimatization during transitions of
animals between different osmotic environments. Special attention is paid to hypotheses about
the diversity of cellular organization of osmoregulatory and excretory organs such as glomerular
kidneys, antennal glands, Malpighian tubules and insect gut, gills, integument and intestine, with
accounts on experimental approaches and methods applied in the studies. It is demonstrated how
knowledge in these areas of comparative physiology has expanded considerably during the last
two decades, bridging seminal classical works with studies based on new approaches at all levels
of anatomical and functional organization. A number of as yet partially unanswered questions
are emphasized, some of which are about how water and solute exchange mechanisms at lower
levels are integrated for regulating whole body extracellular water volume and ion homeostasis
C 2014 American Physiological Society. Compr Physiol
of animals in their natural habitats. 4:405-573, 2014.
Introduction
In his lecture series as professor of comparative physiology
at the Museum of Natural History in Paris Claude Bernard
(1813-1878) wrote: “I believe I was the first to insist on the
concept that animals have actually two milieus; an external
milieu in which the organism is located and an internal milieu
in which the elements of its tissues live. The real existence
of a living thing does not take place in the external milieu,
which is the atmosphere for air breathing creatures and fresh
or salt water for aquatic animals, but in the liquid internal
milieu formed by the circulating organic fluid surrounding
and bathing all the anatomical elements of the tissue” (113).
Inspired by this idea of Bernard and his own studies of glucose
metabolism, body temperature and acid-base balance, Walter
B. Cannon (1871-1945) developed the concept of “homeostasis.” Homeostasis, Cannon pointed out, would require mechanisms operating simultaneously or successively in such a
way that perturbation of a physiological variable initiates processes that would counter the change (250). In experiments
with freshwater animals, August Krogh (1874-1949) discovered that their osmoregulation depends on active ion transport
across the skin and gills and concluded that the different ion
composition of the extracellular and intracellular body fluids,
and of freshwater animals and their natural surroundings, “has
not to do with a true equilibrium, but with a steady state maintained against a passive diffusion and requiring expenditure
of energy” (974).
Volume 4, April 2014
The ideas of the above seminal works are embedded in
the comparative physiology of osmoregulation and excretion
dealing with the regulation of composition and volume of the
internal milieu of multicellular organisms exposed to different and time-varying external milieus. In order to study the
regulation of fluxes of solutes and water between the animal
and the surroundings, the transport systems and their energy
requirement have to be identified and related to the anatomical and cellular organization of ion- and water-transporting
tissues and excretory organs, which have constituted major
efforts in comparative studies of osmoregulation and
excretion.
* Correspondence
to ehlarsen@bio.ku.dk
of Biology, the August Krogh Centre, University of
Copenhagen, Copenhagen, Denmark
2 Biology Department, University of Louisiana at Lafayette, Lafayette,
Louisiana
3 Department of Biological Sciences, Wagner College, Staten Island,
New York
4 Department of Biology, McMaster University, Hamilton, Ontario,
Canada
5 RSMAS, University of Miami, Miami, Forida
6 Department of Physiology, University of Arizona, Tucson, Arizona
7 Department of Biological Sciences, University of Manitoba,
Winnipeg, Manitoba, Canada
Published online, April 2014 (comprehensivephysiology.com)
DOI: 10.1002/cphy.c130004
C American Physiological Society.
Copyright 1 Department
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The present article is organized as follows. The initial section on “Biophysical Concepts in Osmoregulation” presents
commonly used concepts and theoretical tools for the study
of water and ion exchange in animals. Some mathematics is
necessary in order to express the issue in a precisely defined
form. This is done here by explaining the results in qualitative,
biologically relevant terms of importance for animal osmoregulation. The subsequent sections are organized according to
taxonomic groups to emphasize that comparative physiology
of osmoregulation is about principles and diversity of adaptations that have evolved among different animal species to
cope with changing osmotic environments. Our discussions
include physiological and biochemical mechanisms at all levels of functional organization. It will be clear that scientific
progress in different taxa proceeds along somewhat different
paths. We aim at a review on the experimental background of
the present ideas and at providing stepping-stones for future
studies of this major and expanding field of comparative physiology. Specifically, the future challenge is to integrate whole
body, isolated tissue, and cell physiological research with
progress in studies on the organization of the genome and
expression patterns.
Biophysical Concepts
in Osmoregulation
The section summarizes quantitative treatments of water and
ion transport across plasma membranes and epithelia, which
are pertinent to studies and discussions of animal osmoregulation.
Chemical Potential of Water, Osmotic Pressure,
and the van’t Hoff Equation
Assume two compartments of water, (a) and (b), separated
by a water-permeable membrane. The chemical potentials of
(b)
water are given the symbols, μ(a)
w andμw . If water is not in
(a)
(b)
thermodynamic equilibrium (μw = μw ), there is a (net) flux
of water, Jw(net) , directed from the compartment of the higher
chemical potential to that of the lower chemical potential. If
there is no interaction in the membrane between the water
molecules or between water molecules and other moving particles, the flux-ratio equation applies (1889),
RT ln
Jw(net)
Jw(a)→(b)
μ(a)
w
μ(b)
w
=
−
Jw(a)←(b)
= Jw(a)→(b) − Jw(a)←(b)
(1a)
(1b)
where R is the universal gas constant (8.31 J·K−1 ·mol−1 or
0.082 l·atm·K−1 ·mol−1 ) and T the absolute temperature (K).
The unidirectional water fluxes (Jw(a)→(b) , Jw(a)←(b) ) are measured by the isotope tracer technique using 3 H2 O, D2 O or
406
H2 O18 , or by a combination of the tracer technique for determining one of the unidirectional fluxes and a volumetric measurement of the net flux of water. Water transport across biological membranes takes place in the lipid bilayer between
integral proteins (532), via aquaporins constituting selective
water-permeable pores (7, 8, 1483, 1484), and in the narrow
tight junctions of the paracellular pathway of absorbing and
secreting epithelia (906, 1892, 1984). Although the transport
is passive, Eq. (1a) is rarely obeyed because bulk water flow
in pores violates the assumption of random thermal movement of individual water molecules, which was discovered
already in the early studies of water transport in biological
systems (746, 955, 1399). Since then, it has been a major
challenge for physiologists to develop a consistent theoretical
framework for handling of water transport across biological
membranes (532, 804).
The chemical potential of water is determined by the mole
fraction of water (Xw ) according to (1761),
μw = μwO (T, p) + RT ln X w
where μwO (T, p) is the chemical potential of pure water
(Xw = 1) at temperature, T, and pressure, p. Assume that
compartment (a) contains pure water and is separated from
compartment (b) of similar temperature, but containing the
impermeable solute, s of mole fraction Xs . Thus, Xw = 1
of compartment (a), and Xw + Xs = 1 of compartment (b).
(b)
Thermodynamic equilibrium for water, μ(a)
w = μw , requires,
μwO (T, p (a) ) = μwO (T, p (b) ) + RT ln X w
Since Xw < 1, RTlnXw is negative. Thus, p(b) − p(a) > 1, which
is the osmotic pressure, , of the solution in compartment (b)
given by the following thermodynamically exact expression
(1761),
= −
RT
Vw
ln(1 − X s )
(2)
where V w is the mean value of the molar water volume, V w ,
in the pressure range p(a) to p(b) . For a dilute solution, Xs 1
and ln(1 − Xs ) ≈ −Xs that leads to,
= RT
Vw
Xs
(3a)
Introducing the concentration of the solute rather than its
mole fraction presupposes further mathematical approximations. Since Eq. (3a) assumes the solution to be dilute,
Xs = ns /(nw + ns ) ≈ ns /nw , where ns and nw are the number of gram molecules of s and w. Thus,
RT
ns
nw V w
ns
= RT
[Vw ]
=
(3b)
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Comprehensive Physiology
Osmoregulation and Excretion
Here, n w V w = [Vw ] is the volume occupied by water
of
the solution with a total volume of V. Therefore, n s [Vw ]
is the volume molality, which for diluted solutions is equal
to the weight molality, m, in moles per kilogram of water
and referred to as the osmolality of the fluid. The molar concentration or the osmolarity of the solution
is defined by,
cs = ns /V, and for diluted solutions, n w V w ≈ V, which,
inserted into Eq. (3b), gives,
= RT cs
(3c)
This equation expresses the van’t Hoff’s law (1895), with cs
being the solute concentration in moles per liter of solution.
Because the volume of water is temperature dependent, the
osmolarity of a solution is temperature dependent.
Estimation of osmolality of animal body fluids,
colligative properties
Determination of the chemical potential of water is a prerequisite for investigating the nature of the distribution and fluxes
of water between compartments in an animal and between
an aquatic animal and its environment. As indicated above, in
ideal (i.e., diluted) solutions, the osmotic pressure depends on
the number of dissolved particles per unit of volume of water,
but is independent of the chemical composition, size, shape,
and electrical charge. A corollary to this assumption is that the
forces of interactions between water molecules and between
water molecules and dissolved molecules do not differ. For
diluted solutions, the osmotic pressure (Eq. 2) and the osmolality (Eq. 3b) are both measures of the chemical potential of
water. The osmotic pressure is one of four colligative properties. With reference to pure water, the impacts of solutes
on colligative properties are: (i) increase in osmotic pressure,
(ii) lowering of vapor pressure, (iii) elevation of boiling point,
and (iv) depression of freezing point. Eq. (2) and Eqs. (3a-c)
are valid only for dilute solutions. Biological fluids are not
ideal solutions. Because there is no thermodynamically exact
way of dealing with this, if the molar concentration is known,
one may introduce an empirical dimensionless temperaturedependent osmotic coefficient (φ) as correction factor to the
van’t Hoff equation,
= φ RT cs
(3d)
Values of φ for electrolyte solutions of different concentrations are listed in ref. (1567); as an example, for cNaCl =
100 mM at 25 ◦ C, φ = 0.932, and because NaCl in aqueous
solution is fully dissociated, = 0.932·0.0821·298·0.2 =
4.56 atm. Thus, as a first approximation, by adding NaCl
to pure water to a final concentration of 100 mmol/l, the
chemical potential of water would be lowered by μw =
0.932·8.31·298·0.2 = 463 J/l. Generally, the composition of
biological fluids is not known exactly. Therefore, one of the
colligative properties is measured from which the osmolality
is estimated. The freezing point depression and the lowering
Volume 4, April 2014
of vapor pressure can be measured in samples of the order of
10−7 -10−6 l by commercially available instruments and are
therefore preferred in studies of biological fluids. Theoretical
and technical problems in using of these methods to non-ideal
solutions are discussed in ref. (1795).
Transport equations
The volume flow across a membrane that is permeable both
to water and solute is given by (532),
JV = L p (p − σ RT cs )
(4a)
where Lp is the hydraulic conductance, p is the hydrostatic
pressure difference between the two compartments, cs is the
solute concentration difference between the compartments,
and σ is the dimensionless reflection coefficient defined such
that σ = 0 if the membrane cannot distinguish between water
and solute, and σ = 1 if only water can pass. The hydraulic
conductance is related to the osmotic permeability, Pf , by
(532),
Pf =
RT
Vw
Lp
where V w is the partial molar volume of water, which is
∼ 18 cm3 ·mol−1 at 20 ◦ C. In animal cells with soft plasma
membranes, p ∼
= 0 in Eq. (4a). Thus,
JV = P f V w σ cs
(4b)
If the water exchange is studied using isotopes, diffusion
permeabilities (Pdw ) are obtained,
−→
Jw(a→b) = Pdw c(a)
H2 O
←− (b)
(a←b)
Jw
= Pdw c H2 O
For an oil membrane in which the concentration of water
is so low that interactions between water molecules can be
neglected,
Pf
−→ ←−
Pdw = Pdw = Pdw and
=1
Pdw
If there is a net water movement across a porous membrane,
the isotope flux is accelerated in the direction of net water flow
−→
and impeded in the opposite direction, resulting in, Pdw =
←−
Pdw = P f .
This inequality was indicated in the very first study using
isotopes for investigating the nature of water transport across
biological membranes (746).
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Partition of Ions between Intra- and
Extracellular Compartments
Exchange of water, ions, and organic molecules between the
organism and the environment takes place through epithelia of
the integument and the gills, and through “internal” epithelia
such as gastrointestinal tract, kidney tubules, urinary bladder, and exocrine glands. The physical-chemical concepts of
ion transport between extracellular fluid and the surroundings
are the same as those of the exchange of ions between the
cell and the extracellular fluid. The common electrolytes of
body fluids are always fully dissociated into ions. In highly
diluted solutions, the ions move freely without interacting
with each other, so that the mass concentration, c, of an ion
is the same as its activity, a. Biological fluids are not highly
diluted, which means that the activity of an ion on which
its kinetic and thermodynamic properties depend is different
from its mass concentration, a = f·c, 0 < f ≤ 1. The activity
coefficient, f, depends on the valence of the ion, the ionic
strength of the solution, and the dielectric constant of the solvent. The textbook by Robinson and Stokes (1567), which is
frequently consulted by physiologists, discusses theories of
ion-ion and ion-water interactions in fluids of different concentrations and provides tables of activity coefficients and
other thermodynamic parameters of relevance for biological
fluids.
Fig. 1A shows a plasma membrane that separates an
intracellular from an extracellular compartment that contain
the membrane permeable ion, j. Temperature and hydrostatic
(A)
(B)
pressure, respectively, are assumed similar in the two compartments. The ion flux J is defined as the number of moles
of j, which pass the unit area of plasma membrane per unit
time. If the ion transport (or the transport of any solute) is
estimated from the flux of a radioactive isotope, one denotes
the isotope flux from the outside into the cellular fluid as the
‘influx’,J j(in) , while the isotope flux in the opposite direction
is denoted the ‘efflux’, J j(out) . Having accounted for the specific activity of the isotope in the compartment into which it
was introduced, under physiologic stationary conditions the
“net flux” is calculated as, J j(net) = J j(in) − J j(out) .
Electrochemical potentials and
thermodynamic equilibrium
The electrochemical potentials of j, μ̃ j , in the two fluid compartments are:
(e)
O
(e)
μ̃(e)
j = μ + RT ln a j + z j Fψ
(e)
= μ O + RT ln( f j(e) c(e)
j ) + z j Fψ
(c)
O
(c)
μ̃(c)
j = μ + RT ln a j + z j Fψ
(c)
= μ O + RT ln( f j(c) c(c)
j ) + z j Fψ
(5a)
(5b)
where μ◦ is chemical potential at standard conditions, F is the
Faraday (96485 C·mol−1 ), and zj is the valence of the ion. If
Environmental
or luminal
fluid
Tight junction
Extracellular
fluid
Epithelial
cell layer
Basal
lamina
Lateral plasma
membrane
Extracellular
Cellular
Plasma
fluid
fluid
membrane
aic =
fic . cic,
aie =
fie . cie,
ψc
ψe
Apical or
luminal plasma
membrane
Basal plasma
membrane
Translateral
transport
Transcellular
transport
Transjunctional
transport
Figure 1
(A) Plasma membrane (m) separating the cell compartment (c) from the interstitial fluid
compartment (e). Symbols: ψ (c) , ψ (e) : electrical potential of cellular (c) and extracellular fluid (e);
(c) (e)
(c) (e)
(c)
a j , a j : chemical activities of j; c j , c j : chemical concentrations of j; f j , f je : activity coefficients of j.
(B) Transepithelial pathways in epithelia.
408
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Comprehensive Physiology
Osmoregulation and Excretion
(e)
the electrochemical potentials are identical,μ̃(c)
j = μ̃ j , j is in
thermodynamic equilibrium across the plasma membrane,
(c)
(e)
= μ O + RT ln a (e)
μ O + RT ln a (c)
j + z j Fψ
j + z j Fψ
which leads to:
ψ (c) − ψ (e) =
a (e)
RT
j
ln (c)
z j F aj
At electrochemical equilibrium the potential difference,
ψ (c) − ψ (e) , is denoted as j’s equilibrium potential, Ej , at
the prevailing ion activities,
Ej =
≈
a (e)
RT
j
ln (c)
z j F aj
cej
RT
ln c
z j F cj
Vm =
(6)
Membrane potentials: The homogenous membrane
In a multi-ion system, membrane potential, Vm = ψ (c) − ψ (e) ,
depends on permeability and distribution of all membranepermeable ions. While the Nernst equation for the equilibrium distribution of a single ion (Eq. 6) is independent of
membrane structure, the mathematical equation for Vm for the
general non-equilibrium case can only be derived for simplifying assumptions about the membrane’s physical properties.
(e)
(c)
PNa cNa
+ PK c(e)
RT
K + PCl cCl
ln
(c)
(e)
F
PNa cNa
+ PK c(c)
K + PCl cCl
(7a)
It is tacitly assumed that active Cl− transport mechanisms
are non-rheogenic, and that the contribution of the rheogenic
Na+ /K+ pump to the membrane potential can be neglected.
A corollary to the last mentioned assumption is that the specific plasma membrane resistance, Rm , is low. Otherwise, the
pump’s contribution to the membrane potential, Vpump , cannot be neglected, Vpump = Rm Ipump , where Ipump is the pump
current. At steady state with a cation stoichiometry of the
pump
Na+ /K+ pump of β = 3/2, i.e., I pump = F(β − 1) · JK ,
Eq. (7a) reads:
Vm =
Because the ionic strengths of the cellular and extracellular fluids are about the same, as a good approximation,
f j(c) ≈ f j(e) , whereby concentrations of j can replace the activities as indicated in Eq. (6), which is the Nernst Equation.
(c)
Thus, given the concentrations, c(e)
j and c j , Ej is the electrical
potential difference required between the two compartments
for j being in electrochemical equilibrium. Fig. (1B) shows an
epithelium of polarized cells with tight junctions between the
cells separating the extracellular fluid from the environmental
or the luminal fluid. As indicated, there are several anatomical pathways for exchange of ions and water between the
extracellular fluid and the fluid bathing the outside or luminal
aspect of the epithelium. The above thermodynamic equilibrium condition applies as well to the ion distribution across an
epithelium no matter which and how many of the pathways
are involved in the transepithelial transport. For transepithe(c)
(c) (c)
(c)
are
lial transport, the symbols μ̃(c)
j , f j , a j , c j and ψ
(o)
(o)
(o) (o)
(o)
replaced by the symbols μ̃ j , f j , a j , c j and ψ with the
new superscript, (o), referring to the outside (environmental
or luminal) fluid.
Volume 4, April 2014
The Goldman potential equation was derived for a homogenous membrane (see below) for the case Vm is a pure diffusion
potential governed by the three major diffusible ions (775),
(e)
(c)
+ β PK c(e)
PNa cNa
RT
K + PCl cCl
ln
(c)
(e)
F
PNa cNa
+ β PK c(c)
K + PCl cCl
(7b)
In the above equations, the permeability coefficient, Pj is
defined as
Pj =
αj Dj
h
(8)
where α j is the dimensionless partition coefficient relating
the ion’s concentration in the solution to its concentration just
inside the membrane boundary, Dj is the diffusion coefficient
in the membrane, and h is the membrane thickness. If Dj is
in cm2 ·s−1 and h is in cm, Pj is in cm·s−1 . The homogenous membrane fulfils the following requirements: (i) Dj is
independent of the position (x) in the membrane; (ii) the membrane is symmetric, which means that α j at the intracellular
membrane-fluid boundary (x = 0) is the same as α j at extracellular membrane-fluid boundary (x = h); (iii) the electrical
field in the membrane is constant, which means that the electrical potential is a linear function of x; and (iv) the potentials
at x = 0 and x = h are equal to the potentials in the cellular
and extracellular fluids, respectively.
Transport Equations
Diffusion
Net transport by diffusion is a random thermal movement
of molecules of a non-uniform distribution in a solution or
between compartments. Thus, if there is a concentration difference across the membrane of a non-charged solute, there
will be a net transport in direction from the compartment of
higher concentration to that of lower concentration, which is
governed by Fick’s first law of diffusion in one dimension
along the x-coordinate,
Js = −Ds
dcs
dx
(9a)
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The diffusion coefficient, Ds , and the concentration gradient,
dcs /dx, refer to the same arbitrary position along the transport path in the membrane. For a homogenous membrane,
integration of Eq. (9a) leads to
Js = Ps (cs(c) − cs(e) )
(9b)
The permeability coefficient, Ps , is related to the diffusion
coefficient in the membrane by Eq. (8) above. Equation (9b)
follows the convention that a flux directed from the cell to
the extracellular fluid is given a positive sign. For a membrane composed of layers of different thicknesses and diffusion coefficients, which may include external unstirred layers,
P in Eq. (9b) is replaced by an equivalent permeability, P,
which is given by the permeability of the individual layers
according to 1/ P = 1/P1 + 1/P2 + . . . 1/Pn (804, 1761).
Electrodiffusion
An electrical field, dψ/dx, superimposed on the random movement of ions in the membrane expands Eq. (9a) to
J j = −D j
dc j
z j Fc j dψ
+ Dj
dx
RT d x
(10a)
For the homogenous membrane, integration of Eq. (10a) leads
to the Goldman flux equation (Eq. 10b) and the Goldman
current equation (Eq. 10c), respectively (775, 1761),
z j F Vm
J j = Pj
RT
of the water flow that drives the solute from one side of the
pore to the other (27). With v being the convection velocity
(cm·s−1 ) in the pore and Ds the diffusion coefficient of the
solute in the pore, the extension of Eq. (9a) reads:
(c)
c(e)
j − c j exp z j F Vm /(RT )
1 − exp z j F Vm /(RT )
Js = −Ds
I j = Pj
z 2j F 2 Vm
RT
Js = v
cs∗(e) − cs∗(c) exp(vh/Ds )
1 − exp(vh/Ds )
(c)
c(e)
j − c j exp z j F Vm /(RT )
1 − exp z j F Vm /(RT )
(10c)
where, Ij = zj FJj , Pj is given by Eq. (8) and Vm = ψ (c) −
(c)
ψ (e) . For c(e)
j = c j , the current-voltage relationship given by
Eq. (10c) is non-linear, which is referred to as GoldmanHodgkin-Katz rectification (GHK rectification). In characterizing the molecular phenotype of ion channels, Eq. (10c) is a
useful reference. For example, the epithelial sodium channel
(ENaC), which is expressed in many Na+ -absorbing epithelia,
exhibits GHK rectification (562, 1389, 1390, 1899). Singlechannel studies of another important epithelial ion channel,
the CFTR chloride channel, indicated that under certain conditions, the channel exhibits a more complicated Cl− current
rectification than Eq. (10c) predicts (1069, 1796).
Convection superimposed on diffusion: Solvent drag
In fluid-transporting epithelia, water transport through waterand solute-permeable pores acts as a force in the direction
410
(11b)
The symbols cs∗(c) and cs∗(e) refer to concentrations in the pore
at x = 0 and x = h, respectively. Considering 1 cm2 of crosssectional pore area, the convection velocity in cm·s−1 is a
measure of the volume flow per cm2 of uniform membrane
pore, JV (in cm3 ·cm−2 ·s−1 ). Introducing P by Eq. (8), the
reflection coefficient, σ , and the relationship for a symmetrical
pore, α = 1 − σ (532), Eq. (11b) becomes
Js = [(1 − σ )Jv ]
cs(e) − cs(c) exp {(1 − σ )JV /Ps }
1 − exp {(1 − σ )JV /Ps }
(11c)
For an ion that is transported under the influence of a water
flow and an electrical field, as for example at tight junctions, Eq. (9a) is expanded to Eq. (11d), which integrates to
Eq. (11e) below (1005):
J j = −D j
(11a)
Integration through a homogenous pore of uniform crosssection area gives Hertz’s equation (1761),
(10b)
dcs
+ vcs
dx
dc j
z j Fc j dψ
+ Dj
+vcs
dx
RT d x
(11d)
z j F Vm
+ (1 − σ )Jv
J j = Pj
RT
(c)
c(e)
j − c j exp z j F Vm /(RT ) exp (1 − σ )JV /P j
×
1 − exp z j F Vm /(RT ) exp (1 − σ )JV /P j
(11e)
The flux-ratio equation
Equations 9-11 depend on the assumption that the transport
pathway is homogenous as defined above, which is not fulfilled in multi-membrane systems like epithelia. If the transport across a barrier of arbitrary complexity, such as an epithelium, takes place entirely as a consequence of differences in
concentration and electrical potential between the two sides
of the barrier, and if j is not interacting with other moving
particles, the flux-ratio equation is obeyed (1889):
RT ln
J j(in)
J j(out)
(i)
= μ̃(o)
j − μ̃ j
(12a)
J j(net) = J j(in) − J j(out)
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Comprehensive Physiology
Osmoregulation and Excretion
Thermodynamic work of ion pumps
Insertion of Eqs. (5a) and (5b) into Eq. (12a) yields
RT ln
J j(in)
J j(out)
= RT ln
a (o)
j
a (i)
j
+ z j F(ψ
(o)
− ψ ) (12b)
(i)
which can be rearranged to give
J j(in)
J j(out)
J j(in)
J j(out)
=
a (o)
j
a (e)
j
= exp
z j F VT
RT
(12c)
z j F(VT − E j )
RT
(12d)
exp
where VT = ψ (o) − ψ (e) is the electrical potential difference
across the epithelium, and Ej in Eq. (12d) is the equilibrium potential (Eq. 6). A straightforward way of proving this
classical and important equation is presented in ref. (1761).
Thus, a flux ratio analysis constitutes a powerful method of
identifying the mechanism of transport of an ion in question.
The influx and the efflux are measured by means of radioactive isotopes of the ion, or by isotope tracer technique for
measuring one of the unidirectional fluxes combined with
chemical determination of the net transport. Knowing the ion
activities (or the ion concentrations if f j(o) = f j(i) ) and the
trans-epithelial potential difference, by means of Eq. (12d) it
is revealed whether the transport is simple passive (954).
Active Transport: Free Energy Change
of ATP Hydrolysis
Active transport occurs when the flux is coupled with an
exergonic process at the plasma membrane, which may be
hydrolysis of ATP (primary active transport) or a passive
flux of another ion (secondary active transport) transported
either in the same direction of j (cotransport) or in the opposite direction of j (counter transport). The hydrolysis of ATP
proceeds as follows,
+
ATP4− + H2 O → ADP3− + HPO2−
4 +H ,
with the change in free energy associated with the hydrolysis
of 1 mol ATP given by
G (c)
= G O + RT ln
ATP4−
(c)
(c)
cADP
3− c
HPO2−
4
(c)
cATP
4−
3Na+(c) + 2K +(e) + ATP4− + H2 O(c) → 3Na+(e) + 2K +(c)
+
+ADP3− + HPO2−
4 + H
In osmoregulatory epithelia, the 3 Na+ are taken up across the
apical plasma membrane, while the 2 K+ recycle across the
basolateral plasma membrane (956, 1312). Thus, the theoretical maximum work that can be done in transporting 1 mol of
/3 ≈ 60/3 = 20 kJ.
Na+ across the epithelium is −G (c)
ATP4−
A single cycle of the H+ pump ATPase leads to secretion of
2 H+ to the outside fluid at the expense of the hydrolysis of
1 ATP4− together with the exchange of 2 Cl− in the outside
bath with 2 HCO3 − in the cellular fluid,
2H +(c) + 2HCO−(c)
+ 2Cl−(o) + ATP4−(c) + H2 O(c) →
3
2H +(o) + 2HCO−(o)
+ 2Cl−(c) + ADP3−(c) + HPO2−(c)
3
4
+ H +(c)
Subsequently, 2 Cl− are passively transported across the basolateral membrane to the extracellular fluid. In this case, the
theoretical maximum work that can be done in transporting
/2 ≈ 60/2 =
1 mol Cl− across the epithelium is −G (c)
ATP4−
30 kJ. With reference to Eqs. (5a) and (5b) above, and with
superscript (o) for freshwater, the electrochemical work done
in transporting 1 mol of Na+ and 1 mol of Cl− from freshwater
to the extracellular fluid as a first approximation is
μ̃Na + μ̃Cl = RT ln
(e)
aNa
(o)
aNa
+RT ln
, pH(c) = 7.0
where G o = G o + RT ln cH(c)+ = −30.5 kJ · mol−1 is the
change in standard free energy at 25 ◦ C at 1 M concentration
of reactants and products, at cH(c)+ = 10−7 M. At typical cellular concentrations, G (c)
≈ −60 kJ · mol−1 . This would
ATP4−
be the theoretical upper-limit electrochemical work of an ion
pump energized by ATP hydrolysis, noting that 100% energetic coupling efficiency cannot be achieved.
Volume 4, April 2014
In the context of osmoregulation of freshwater animals, the
thermodynamic work associated with branchial or integumental transcellular uptake of Na+ and Cl− is of particular interest.
These osmoregulatory and respiratory tissues express the Ptype Na+ /K+ pump ATPase at the (baso)lateral plasma membrane (858, 1257, 1275, 1545, 1709, 1710) and the V-type H+
pump ATPase at the apical plasma membrane (922, 945, 1058,
1062, 1303, 1412, 1848, 1949). At physiological conditions
one cycle of the Na+ /K+ pump results in translocation of 3
Na+ from the cellular to the extracellular fluid and 2 K+ in
the opposite direction at the expense of hydrolysis of 1 ATP4−
to 1 ADP3− and 1 HPO4 2− ,
= RT ln
+ F VT
(e)
aCl
(o)
aCl
− F VT
(e) (e)
cNa
f Na
(o) (o)
f Na
cNa
+ RT ln
(13)
(e)
f Cl(e) cCl
(o)
f Cl(o) cCl
Energy expenditure of ion uptake from
diluted solutions
Assume extracellular concentrations of Na+ and Cl− of
125 mM, and a ratio of activity coefficients of 0.78; if only
the 3Na+ /2K+ pump ATPase is involved and the Cl− uptake is
411
Osmoregulation and Excretion
passive and driven by the transepithelial potential, the uptake
of the two ions would cease already at an external concentration of Na+ and Cl− between 5 and 1 mM (μ̃Na + μ̃Cl ≈
15 and 23 kJ, respectively). This is well above the ion concentrations of a majority of freshwater pools. With the additional contribution of the H+ ATPase (846, 848, 1371, 1379,
1424, 1425), whereby −(G (c)
/3 + G (c)
/2) ≈ 50 kJ,
ATP4−
ATP4−
+
the uptake of 1 mol Na and 1 mol Cl− would be possible
at about 10 μM (μ̃Na + μ̃Cl ≈ 45 kJ). In his study published in 1937, Krogh observed that Cl− is taken up by a
salt-depleted frog immersed in solutions down to about 10
μM provided Na+ constituted the matching cation (978). If
the apical anion exchanger is rheogenic with a stoichiometry
of Cl− /nHCO3 − (n > 1) and still coupled to an apical H+
ATPase, it would be possible to take up Na+ and Cl− at even
more diluted environments (657). For example, if n = 2, two
protons are pumped out of the cell for each Cl− taken up via
the anion exchanger thus increasing the ATP: Cl− stoichiometry from 0.5 to 1. Therefore, the theoretical maximum work
in transporting 1 mol of Na+ and 1 mol Cl− becomes 80 kJ
rather than 50 kJ, which would make transepithelial uptake
of the two ions thermodynamically possible at environmental
concentrations of Na+ and Cl− in the order of 100 nM.
The above examples illustrate principles of energy transfer
in active ion transport. In a given situation, temperature and
concentrations of the participating ions and metabolites have
to be considered. If energetically feasible, whether uptake
of Na+ and Cl− does take place depends on the affinity of
the ion-binding sites of the transport systems. The zebrafish
(Danio rerio) has the capacity for increasing the affinity of
its branchial transport systems upon acclimation to micromolar Na+ and Cl− concentrations (150), which indicates
that this species would be able to exploit the power of the
above combination of transport ATPases.
Solute-Coupled Water Transport and
Isotonic Transport
Early studies of water transport by kidney proximal tubule
(1618), small intestine (364) and gallbladder (423) revealed
a sizable transepithelial fluid flow at transepithelial osmotic
equilibrium of an osmolality similar to that of the bathing
solutions. The rate of fluid transport was proportional with
the active flux of Na+ . A similar “isotonic transport” has
been observed for the acinar epithelium of exocrine glands
(1819, 1820), Malpighian tubules (1998) and amphibian skin
epithelium (1313, 1314). For fluid-absorbing epithelia it was
suggested that such a flow of water in the absence of a
transepithelial osmotic pressure difference proceeds in two
steps (363, 1982). Apically, transport of water into the lateral space is forced by a small osmotic pressure difference
between the luminal (external) solution and the lateral intercellular space. Subsequently, water exits across the interspace
basement membrane of a reflection coefficient near zero, thus
being forced by the hydrostatic pressure difference between
the lateral space and the interstitial space (conf. Eq. 4a). In
412
Comprehensive Physiology
entering the epithelium, water may take a transjunctional
and/or a translateral route into the lateral space (defined in
Fig. 1B), with additional solute-water couplings in the lumen
of basolateral infoldings (1866). This hypothesis is supported
by the finding that sodium pumps in transporting epithelia
are expressed preferentially or exclusively at the lateral membranes and at the membranes of basolateral infoldings (499,
658, 886, 1228, 1229, 1457, 1770). With no further assumptions, in principle this hypothesis accounts for uphill water
transport as well (998, 1961). It is not immediately obvious, however, how the transported fluid becomes isotonic
at transepithelial equilibrium, because the fluid flowing out
of the lateral space through the interspace basement membrane would be hypertonic. Careful experimental analysis has
shown that the osmotic water permeability of some but not all
transporting epithelia is very high, resulting in a tonicity of the
transported fluid that was estimated to be only slightly hypertonic. Thus, it was suggested to replace the concept of isotonic
transport with that of “nearly isotonic transport” (1747).
Theories of truly isotonic transport
Theories have been developed that handle “truly isotonic
transport.” In the Diamond-Bossert standing gradient theory
(424), it was assumed that tight junctions are water impermeable and sodium pumps are concentrated at the lateral
membranes near the luminal end, so this region of the lateral space becomes sufficiently hypertonic for driving water
into the space from the luminal solution via cells. Next, the
osmotic pressure gradient is being dissipated as water flows
into the lateral space from the cells (see Fig. 2A). Mathematical analysis (424) showed that by appropriate combination of
the transport variables osmotic equilibrium can be achieved
at the boundary between the lateral space and the interstitial space. Diamond and Bossert rationalized their theory by
pointing out that diffusion of solutes out of the lateral space
would be very large as compared to the convection term even
in the presence of a small difference in solute concentration
between the lateral fluid and the interstitium, which would
result in a strongly hypertonic fluid emerging from the lateral
space (424). This pertinent feature of a convection-diffusion
process at a membrane of high solute diffusion permeability
is recognized by function analysis of Eq. (11c) (997, 1006).
In the Na+ recirculation theory (Ussing), it is acknowledged that the fluid emerging from osmotic coupling compartments is hypertonic and assumed that simultaneous stationary
fluxes return Na+ (and Cl− ) into the coupling compartments
from the interstitial space via the cells (1005, 1009, 1301)
(see Fig. 2B). According to this theory, isotonic transport is
achieved by a regulated balance between a forward flow of
ions and water from the lateral space and the lumen of basal
infoldings, respectively, into the interstitial fluid, and a backward flow of ions through the cells into the two types of
coupling compartments (Fig. 2B), denoted by Ussing as “ion
recirculation.” At steady state, the entrance of water across
the apical barriers is driven by the osmotic pressure difference
Volume 4, April 2014
Comprehensive Physiology
Osmoregulation and Excretion
experimental (1599) and theoretical (1584) studies have led
to the suggestion that electro-osmotic coupling at tight junctions represents one of the basic mechanisms driving fluid
transport across some leaky epithelia (534). According to this
theory, secretion of a paracellular fluid flow is driven by a
Na+ current in leaky tight junctions lined by fixed negative
charges. The paracellular Na+ current is assumed energized
by a lumen-negative transepithelial potential generated by
active transcellular ion fluxes (535). Two more theories of
isotonic transport are water transport by a Na+ /glucose transporter (SGLT) in the luminal plasma membrane (1218), and
active ATP hydrolysis-driven water transport at tight junctions (750). With emphasis on experimental support and short
comings, the main ideas of each of the above five theories of
isotonic transport are discussed in refs. (998, 1009).
between the osmotic coupling compartments and the luminal
solution. Thus, at constant pump fluxes (and volume flow),
the osmotic pressure of the lateral space and the lumen of
basal plasma membrane infoldings, respectively, increases
with decreasing hydraulic conductance of the apical barriers,
resulting in an increased diffusion flux out of the osmotic
coupling compartments into the interstitial fluid. It follows
that the demand of solute recirculation for achieving overall
isotonic transport depends critically on the hydraulic conductance of the apical barriers (998, 1006). For comparison, the
assumptions of the standing gradient theory and the Na+ recirculation theory, respectively, are outlined in parallel panels of
Fig. 2. For both theories of passive inflow of water across the
apical cell membrane, it is important to realize that under the
condition of osmotic equilibrium between the luminal (apical) and the interstitial (serosal) space, water is entering the
epithelial cells also across their basal membrane, if this membrane is water permeable. It follows that all of the water exits
the epithelium through the interface between the lateral intercellular space and the basal infoldings, respectively, and the
interstitial space, the mechanism of which is in the focus of
the Na+ recirculation theory.
Quite different theories of driving forces for isotonic
fluid transport are also discussed in the literature. Firstly,
Volume 4, April 2014
Annelida and Mollusca
Molluscs and annelids inhabit a wide variety of habitats,
including the ocean, estuaries and oligohaline seas, freshwater lakes and rivers, hypersaline waters, and land. Each
habitat presents the animals living in it with a different mix
of environmental variables; evolution has produced a broad
diversity of physiological mechanisms involved in salt and
water balance in these groups of animals. The salinity of the
water is an important environmental factor that determines
the diversity of living organisms in aquatic habitats. This relationship is illustrated by the classic curve of Remane (1520)
(Fig. 3).
Species diversity is very high in marine and freshwaters, decreases markedly in brackish waters, and is quite low
in hypersaline habitats. Clearly, the physiology of salt and
water balance is a highly significant factor in the evolution
of diversity among aquatic animals. Many marine invertebrates are osmotic conformers—that is, the osmotic concentration (and usually the ionic composition) of the extracellular
fluid is similar to that of the ambient seawater. In contrast,
all freshwater species are osmotic hyper-regulators, with an
Number of species (arbitrary units)
Figure 2 Theories of truly isotonic transport where all of the water
exits the epithelium through the interface between the lateral intercellular and the interstitial space and through the opening of the basal
plasma membrane infoldings as explained in the the text. (A) In the
standing-gradient theory it is assumed that the lateral Na+ /K+ pumps
are displayed near the tight junction and the water channels in the
lateral plasma membrane along the entire lateral intercellular space.
With appropriate choice of model variables, the condition of zero solute
concentration gradient at the interface between the lateral intercellular
space and serosal bath is obtained (424). (B) The Na+ recirculation
theory. The boundary condition of Fig. 2A is replaced by the assumption that a regulated back flux of ions through the serosal plasma
membrane, driven by the Na+ /K+ pumps, adjusts the composition of
the absorbed fluid to the demanded isotonicity (1301). Note that the
Na+ /K+ pumps play a dual role for isotonic transport. Firstly, the pump
activity maintains the driving force for water uptake from the luminal
solution. Secondly, the pump activity energizes the ion recirculation for
achieving an isotonic net transportate. Modified from (1009).
60
50
40
30
20
10
0
0
500
1000 1500 2000 2500 3000 3500 4000
Environmental osmolality (mosmol/kg)
Figure 3 Number of aquatic species in relation to environmental
salinity. Redrawn from Remane (1520).
413
Osmoregulation and Excretion
Comprehensive Physiology
extracellular fluid osmotic concentration higher than that of
the ambient medium. Most animals that inhabit brackish water
show a combination of these behaviors: the extracellular fluid
conforms to the ambient medium at higher salinities and is
hyperosmotic to the medium at lower salinities. For animals
in terrestrial habitats, the primary factor in the maintenance
of salt and water balance is evaporative water loss to the environment. These animals obtain water by drinking and from
their food. Ions are obtained from the diet.
Marine Osmoconformers
Marine animals that are osmotic conformers may be either
stenohaline (able to survive within a narrow range of environmental salinity) or euryhaline (tolerant of a wide range
of salinity). In these animals, the osmotic concentration of
the body fluids is usually slightly hyperosmotic to the ambient concentration (see [1359, 1681] for examples). The ionic
composition of the hemolymph of osmotic conformers is,
with a few exceptions, nearly identical to the ambient seawater (Table 1). Pelagic animals have reduced concentrations
of sulfate in the body fluids, probably to increase buoyancy.
The urine produced by these animals is iso-osmotic to the
medium (Table 2). Because of wide differences in methodology, the values for urine production rates in Table 2 should
be accepted with caution. Nonetheless, the rates of urine production in some of marine animals are not markedly different from those found in comparable freshwater animals;
Table 1
the reasons for this are unknown. Stenohaline bivalves are
more permeable to water than are intertidal species, but the
permeability of freshwater clams is as high as that of marine
species (1491). An osmotic conformer exposed to a large
decrease in the osmotic concentration of the ambient medium
will take up water by osmosis and lose ions by diffusion.
The magnitude of these diffusive fluxes depends on the scale
of the change in the ambient osmolality. Reductions in the
permeability of the body wall to water have been reported
in both molluscs (406, 1491) and polychaetes (539) exposed
to reduced osmotic concentrations. This mechanism would
slow the rate of water influx and provide the animal with
more time to compensate. The reported decreases in permeability are, however, small in comparison to differences in the
permeability of the body wall between marine and freshwater
animals (941).
Marine osmotic conformers: Volume regulation
For an osmoconformer, any change in the ambient osmotic
concentration results in an osmotic gradient between the
extracellular fluids (and cells) and the surrounding medium.
The process that returns the cytoplasmic and extracellular
fluid compartments to osmotic equilibrium with the ambient
medium is called volume regulation. If the ambient salinity
increases, the animal loses water; a decrease in the ambient salinity results in an uptake of water. In many of these
animals, the initial gains or losses in water are partially or
Osmotic (π , mosmol/kg) and Ionic (mM) Concentrations in Seawater and the Body Fluids of Soft-Bodied Invertebrate Osmotic
Conformers
Seawater
π
Na+
K+
Ca2+
Mg2+
Cl−
SO4 2−
Reference
1,033
480
10
10
55
560
29
(74)
Species
Internal Ratio/External
Aurelia aurita (C)
0.999
0.989
1.058
0.956
0.972
1.038
0.471
(1564)
Phascolosoma vulgare (S)
—
1.038
1.057
1.043
0.686
0.985
0.913
(1563)
Aphrodita aculeata (P)
—
0.994
1.260
1.000
0.988
1.003
0.999
(1359)
Arenicola marina (P)
—
1.001
1.035
0.998
1.003
0.997
0.992
(1359)
Amphitrite brunnea (P)
—
0.976
1.429
1.016
1.100
0.988
1.027
(1359)
Mercierella enigmatica (P)
1.140
0.903
1.670
0.882
1.238
0.941
—
(1359)
Mytilus edulis (B)
—
0.990
1.183
1.135
0.965
0.989
1.004
(1474)
Pecten maximus (B)
—
0.997
1.325
1.017
0.784
0.976
1.029
(717)
Strombus gigas (G)
—
0.998
1.080
1.068
1.074
1.007
0.717
(1079)
Scutus breviculus (G)
1.012
1.008
1.036
1.033
1.025
—
0.987
(1875)
Eledone cirrosa (Cp)
—
0.986
1.540
1.167
1.084
1.008
0.780
(1567)
Sepia officianalis (Cp)
—
0.889
0.991
1.075
1.090
1.085
—
(1)
Octopus dofleini (Cp)
—
0.911
1.132
0.921
—
0.941
0.735
(1477)
C = Colenterate, S = Sipunculid, P = Polychaete, B = Bivalve, G = Gastropod, Cp = Cephalopod
414
Volume 4, April 2014
Comprehensive Physiology
Table 2
Osmoregulation and Excretion
Urine Production in Soft-Bodied Invertebrates
Species
π ext (mosmol/kg)
Urine/hemolymph ratio
Rate (μl g−1 hr−1 )
Reference
Strombus gigas (G)
1,100
0.96
3
(1079)
Haliotis rufescens (G)
1,100
1.0
6-20
(701)
Octopus dofleini (Cp)
1,100
1.0
3
(702)
Nereis diversicolor (P)
1,100
1.0
3
(1720, 1721)
Anodonta cygnea (B)
FW
0.67
12
(1446, 1475)
Margaratana margarantifera (B)
FW
0.09
3-15
(267)
Pomatia limata (G)
FW
0.23
6
(1077)
Viviparus viviparus (G)
FW
0.28
15
(1078)
Hirudo medicinalis (H)
FW
0.10
40
(1965)
Nereis diversicolor (P)
70
0.60
30-130
(1721, 1722)
Lumbricus terrestris (O)
FW
0.19
7
(430, 1504)
Helix pomatia (G)
T
—
10
(1916)
Achatina fulica (G)
T
0.72
4
(1172)
G = Gastropod, Cp = Cephalopod, P = Polychaete, B = Bivalve, H = Hirudinea, O = Oligochaete
completely reversed. The capacity for volume regulation in
marine osmotic conformers varies from extremely limited
(e.g., sipunculids) to very high (some polychaetes and molluscs) (467, 574, 1360, 1447, 1914). Transfer of the sipunculid
Themiste dyscritum from seawater to 50% seawater results in
a 65% increase in the weight of the animal; there is no volume
regulation (1360). The polychaete Arenicola marina is able
to volume regulate completely when transferred from 32
to 20 but retains about 10% of the water gained when the
transfer is to 10 (1519). The bivalve Geukensia demissa
regains volume in 48 hr following a transfer from 36 to
3 (1447). There is no evidence of volume regulation in
G. demissa transferred from 36 to 48 (1447). In both
molluscs and annelids, the responses to hypo- and hyperosmotic media are asymmetrical: volume regulation is more
rapid and more complete following exposure to hypoosmotic
media than following exposure to hyperosmotic media (573,
574, 1362).
The cells of these animals respond to osmotic swelling or
shrinkage by adjustment of the cytoplasmic pool of osmolytes.
The primary inorganic ion in the intracellular osmotic pool is
K+ , but the concentration of potassium ions in the cytoplasm
is always below about 200 mM (941). Since the osmotic concentration of the body fluids (and therefore, the cells) of an
animal that is a conformer at higher salinities can be over 2000
mosmol/kg (1450), the cytoplasm of marine animals contains
high concentrations of organic molecules such as amino acids
and quaternary amines. High concentrations of these compatible solutes are less deleterious to protein structure and function than high concentrations of inorganic ions (2050). The
amino acids that constitute the cytoplasmic pool vary among
species and even among populations of the same species (958,
1449, 1939). The amino acids that are commonly abundant
Volume 4, April 2014
include taurine, alanine and glycine; β-alanine is also abundant in coelenterates (408, 467, 887, 958, 1519). Quaternary
amines include proline betaine and glycine betaine (1448,
1449).
In animals exposed to a decrease in salinity, the osmotic
concentration of the extracellular fluid falls, and the cells
take up water. The cells release cytoplasmic ions and organic
osmolytes into the extracellular fluid to eliminate the osmotic
gradient across the plasma membrane (408, 554). The amino
acids released by the cells are not excreted; most are deaminated by unknown tissues in the animal; the ammonia is
excreted, but the carbon skeletons are presumably conserved
(75, 1149). Exposure of animals to an increase in the ambient
salinity results in loss of water from their cells. Accumulation of cytoplasmic osmolytes reverses this osmotic loss
of water in those species capable of volume regulation. In
bivalve molluscs, the cytoplasmic levels of alanine rise very
quickly following transfer to a hyperosmotic medium; over
time, glycine and taurine accumulate more slowly (60). The
complex changes in amino acid metabolism that are involved
in hyperosmotic volume regulation in bivalves have been studied (142).
The efficacy of volume regulatory mechanisms in
excitable cells in a few euryhaline invertebrates has been
studied in detail. There is variability in the responses of the
neurons of annelids and bivalves, but these cells adjust rather
rapidly to dilute media and can generate action potentials
despite large decreases in the intracellular concentrations of
Na+ and K+ (90, 110, 1999). The isolated ventricles of bivalve
molluscs also recover rapidly from either dilution or concentration of the bathing medium and initiate spontaneous
mechanical activity within a few hours of the change in the
ambient osmotic concentration (1937).
415
Osmoregulation and Excretion
Table 3
Comprehensive Physiology
Aspects of Osmoregulation in Oligohaline and Freshwater Soft-Bodied Invertebrates
π ECF in FW mosmol/kg
π break mosmol/kg
Reference
Oligohaline Animals
Polymesoda caroliniana (B)
48
60
(407)
Mytilopsis leucophaeta (B)
40
70
(410)
Potamopyrgus jenkinsi (G)
125
125
(460)
Assiminea grayana (G)
180
150
(1080)
Enchytraeus albidus (O)
404
400
(593)
Nereis diversicolor (P)
180
335
(1720)
Nereis limnicola (P)
175
350
(1361)
Freshwater Animals
Limnoperna fortunei (B)
40
70
(410)
Lampsilis teres (B)
50
50
(855)
Lymnaea stagnalis (G)
95
100
(404)
Pomacea bridgesi (G)
100
100
(855)
30
30
(537)
145
200
(252)
Craspedacusta sowberyi (C)
Lumbricus terrestris (O)
Animals are osmotic conformers in ambient concentrations above and hyper-regulators below π break .
B = Bivalve, C = Coelenterate, G = Gastropod, O = Oligochaete
Oligohaline and Freshwater Animals
Oligohaline invertebrates that inhabit brackish waters are, for
the most part, osmotic conformers at higher salinities and
hyperosmotic regulators at low salinity. This also applies to
most freshwater species; freshwater molluscs and annelids
have a lesser tolerance for increased ambient salinity than do
brackish water species (408, 1359). The external salinity that
causes the onset of hyper-regulation of the extracellular fluid
varies considerably among these animals (Table 3). In some
animals the permeability of the body wall to ions is reduced
when the animals are regulating the osmotic concentration of
the body fluids (440, 1358). The osmotic concentration of the
hemolymph of freshwater bivalves and coelenterates is lower
than that of freshwater snails and annelids. This reduces the
gradients for passive loss of ions to the medium and uptake of
water by osmosis, but is not reflected in lower rates of urine
production in freshwater bivalves (Table 2). All freshwater
animals produce urine that is hypoosmotic to the body fluids;
however, the ratio of the osmotic concentration of the urine
to that of the hemolymph is lower in freshwater vertebrates
than in freshwater molluscs and annelids (Table 2) (941). To
compensate for diffusive losses of ions and ions lost in the
urine, hyper-regulators must take up ions from the medium by
active transport. In bivalves, the site of uptake of chloride and
sodium is probably the gills; putative transporting cells have
been described in at least one species (431, 907). Worms take
up Na+ and Cl− ions across the body wall (20, 427). The KM ’s
of these ion uptake systems in molluscs and the earthworm
are similar, ranging from 0.05 to 1.5 mM (408, 427, 430). The
416
maximal rates of uptake are also similar in the earthworm (1
μmol/g dry wt/hr) to the lower end of the range of rates for
freshwater molluscs (1-30 μmol/g/hr). The rates of uptake
of Na+ and Cl− by the horse leech Haemopis sanguisuga
in fresh water are about 5 μmol/g dry wt/ hr (976). These
values are consistent with those reported for other freshwater
animals. As in other animals, the uptake mechanisms for Cl−
and Na+ are independent (306, 404, 429, 430, 976). There is
a paucity of specific information on the identity of the proteins involved in the uptake of ions in these animals. The
uptake of sodium by freshwater mussels involves a Na+ -H+
exchanger (428). Chloride uptake in mussels is inhibited by
4,4 -Diisothiocyano-2,2 -stilbenedisulfonic acid (DIDS), suggesting that a chloride exchanger participates in the process
(431). In leeches, integumental uptake of sodium is inhibited
by furosemide and amiloride; this suggests the involvement
of a passive sodium channel and a Na+ -K+ -2Cl− exchanger
(306). In freshwater bivalves, the concentration of bicarbonate in the hemolymph is roughly equal to that of chloride; in
gastropods and annelids, chloride is the predominant anion
(408, 1359). In some leeches, organic acid concentrations in
the hemolymph can total 25 mM (1307).
Terrestrial Animals
For a terrestrial animal with an extracellular fluid osmotic
concentration of 200-400 mosmol/kg, an ambient relative
humidity of less than 99.5% results in evaporative loss of
water. Terrestrial annelids and molluscs dehydrate rapidly in
Volume 4, April 2014
Comprehensive Physiology
Table 4
Osmoregulation and Excretion
Evaporative Water Loss Rates in Terrestrial Soft-Bodied Invertebrates
Species
Air Relative Humidity (%)
Water Loss (μl/g/hr)
Reference
Lumbricus terrestris (O)
70-80
24
(251)
Limax maximus (G)
70-80
40
(1486)
Arion ater (G)
40-80
10
(807)
Helix aspersa (G)
55
45
(1120)
Cepaea nemoralis (G)
65
4
(244)
O = Oligochaete, G = Gastropod
dry air (Table 4). Terrestrial molluscs and annelids can survive the loss of 70%-90% of their total body water (1120,
1485, 1576). Water loss in gastropods without a shell (e.g.,
Limax) is not higher than that of shelled species (e.g., Helix).
Snails can reduce evaporative water loss by two orders of
magnitude by withdrawing into the shell; sealing the shell
aperture with an epiphragm results in a further decrease
of about 50% (1120). During prolonged exposure to desiccation, annelids reduce water loss by estivation inside a
secreted mucus cocoon (475). Dehydrated terrestrial molluscs
and annelids take up water by osmosis across the body wall
when conditions permit (430, 1485). In terrestrial molluscs
subjected to desiccation, the concentrations of organic and
inorganic osmolytes in the cells increase as the osmotic concentration of the extracellular fluids rises (433, 1994).
Urine Formation
In molluscs, the initial stage of urine formation is filtration
of the hemolymph. This occurs in the heart-pericardial organ
complex. In many species, the hydrostatic pressure that drives
filtration is generated by contractions of the heart that drive
hemolymph into the pericardial cavity (540, 745). Podocytes
have been described in tissues such as the auricle, pericardial
gland and kidney in a wide variety of species (44, 45, 714,
1219, 1368, 1622, 1936). The podocytes and associated basal
lamina form a selective filtration barrier; filtered hemolymph
passes from the hemocoel into the pericardial cavity. The filtrate then flows through a short ciliated reno-pericardial canal
to the kidney. The filtrate is processed as it passes through the
kidney; in freshwater animals, ions are reabsorbed to produce
hypoosmotic urine. There is some evidence that some of the
cells in the kidney have a secretory function (918).
In annelids, the excretory organs are nephridia and the
primitive condition is one pair of nephridia per segment. In
many species, the number of nephridia is greatly reduced and
may be limited to a single pair (e.g., fanworms). Podocytes
have been found in association with lateral blood vessels
adjacent to the nephrostome; presumably, filtration of the
hemolymph occurs here (527, 837, 1719). The proximal end
of the nephridium (nephrostome) opens to the coelomic cavity
and the distal end opens to the outside of the animal. Processing of the urine occurs as it passes along the nephridial tubule
Volume 4, April 2014
from nephrostome to the excretory pore. In some leeches, the
nephridia produce urine by secretion rather than filtration; this
is an adaptation that facilitates the rapid excretion of the large
salt and water loads the animals incur by feeding on body
fluids (1965).
Biomineralization
Many species of molluscs produce a calcareous shell and
the serpulid polychaetes surround themselves with calcareous tubes. In marine osmotic conformers, the concentration
of calcium in seawater is the same (10 mM) as that of the
hemolymph. In serpulid worms, formation of the tube requires
the deposition of calcium carbonate into a solution (seawater) saturated with these ions. A gland in the anterior end
of the animal produces calcareous granules suspended in
a matrix that are secreted to the exterior; this material is
molded into shape and then solidifies to form the tube (1694).
Marine molluscs secrete calcium carbonate to form the shell.
In freshwater habitats, calcium concentrations vary between
0.07 and 1 mM depending on the hardness of the water (878).
The concentrations of Ca2+ in the hemolymph of freshwater
molluscs range between 2 and 10 mM (408); shell-bearing
molluscs that inhabit fresh waters must therefore accumulate
large amounts of calcium against a concentration gradient.
In molluscs, the tissue that secretes the shell is the mantle
epithelium. The mechanism responsible for the translocation
of calcium from the hemolymph to the extrapallial fluid is
unknown, but the electrophysiology and the transport of ions
in this tissue suggest that it does not actively transport Ca2+
from the hemolymph to the extrapallial fluid that bathes the
shell (327, 811, 834). These results suggest that calcium ions
are actively transported from the medium into the hemolymph
by active transport at the gill. Measurements of Ca2+ uptake
by intact freshwater clams and snails produce KM values for
uptake that range from 0.1 mM to 0.3 mM; the KM for marine
animals is 7.5 mM (408, 875).
Osmoregulation: Neural and Endocrine Control
The uptake of ions in some freshwater bivalves follows a diurnal rhythm (638, 1200). These observations suggest that the
nervous system regulates the function of the uptake mechanisms. Serotonin and cAMP stimulate the uptake of ions
417
Osmoregulation and Excretion
Crustacea
The Crustacea are a subphylum of the arthropods with approximately 50,000 described species. Crustaceans are predominantly aquatic animals inhabiting seawater, freshwater and
all types of brackish waters. Many species are remarkably
euryhaline, acclimating to extremely different environmental salinities. Members of some crustacean groups, including
crabs, hermit crabs and woodlice, have adapted to a terrestrial
life. Osmotic and ionic regulation in Crustacea has been studied on all levels, from the whole animal to single molecules,
and results have been reviewed on a regular basis. This summary tries to address all major topics of crustacean osmotic
and ionic regulation. For further details, the reader is referred
to the following list of reviews, book chapters and books that
address osmotic and ionic regulation in Crustacea or special
topics of this area (556, 613, 740, 933-935, 941, 1085-1087,
1110, 1151, 1338, 1381, 1418, 1421, 1476, 1811, 1845, 1846,
1848).
Osmotic and Ionic Concentrations
in the Hemolymph
Until the mid-20th century, studies of osmotic and ionic regulation in Crustacea were almost exclusively performed on
whole animals, and even afterward a considerable amount
of important information was collected in this way. The
hemolymph osmolality and ion composition of many crustaceans were determined, and often their changes in response
to exposure to different media were monitored. From this
wealth of data (probably most extensively reviewed in [1151])
a number of conclusions are evident. Groups with clearly
different osmoregulatory capabilities can be distinguished
among the Crustacea (see Fig. 4).
Florkin (541) distinguished two types of osmotic regulation. Intracellular isosmotic regulation occurs in euryhaline
crustaceans that did not evolve the capability to maintain
osmotic gradients across their body surface. The osmoregulatory processes in this group of animals are limited to
the adjustment of the osmotic concentration of all cells to
the osmolality of the hemolymph, which is equal to the
osmotic strength of the ambient medium. In contrast, extracellular anisoosmotic regulation occurs in those crustaceans
that maintain an osmotic gradient across the body surface
by active transport of ions (mainly NaCl) across epithelial tissues. The first group consists of osmoconformers, not
maintaining considerable osmotic gradients across their body
418
1000
800
Hemolymph [mosmol/kg]
and the uptake of sodium ions is inhibited by prostaglandins
in freshwater mussels (434, 638, 802, 1619). Neurosecretory
products alter kidney function in freshwater snails (919). In
leeches, the neuropeptides angiotensin II amide and FMRFamide affect the movement of sodium across the body wall
and control resorption of ions by the kidney (1223, 1965).
Volume regulation in marine molluscs is affected by neuropeptides including FMRFamide (409, 1963).
Comprehensive Physiology
600
400
200
0
0
200
400
600
800
Ambient medium [mosmol/kg]
1000
Figure 4
Osmotic concentration in the hemolymph of different crustaceans in ambient media of different osmotic strength. Isosmotic line
(dotted line), osmoconformers (bold continuous line), weak hyperosmoregulator (dashed line; values for Carcinus maenas, recalculated
after (1660), strong hyperosmoregulators (continuous line; values for
Eriocheir sinensis, after [1372]), hyper-hyposmotic regulator (dotted
and dashed line; Artemia salina, values after [360]).
surface. Thus, these animals do not spend energy for extracellular osmotic regulation. Although many of these animals inhabit marine environments, they can be considerably euryhaline. Migrating between seawater and brackish
waters, their hemolymph osmolality changes considerably. In
response to these changes euryhaline osmoconformers regulate the osmotic concentrations of the body cells, a process
termed intracellular osmoregulation (see below). A considerable number of Crustacea are hyperosmoregulators, maintaining a higher osmotic concentration in their body fluids than
in the aquatic environment. All freshwater species and euryhaline crustaceans that succeed to invade considerably dilute
brackish waters and freshwater are hyperosmoregulators. Two
groups can be distinguished among the hyperosmoregulating,
euryhaline crustaceans that can migrate between seawater and
dilute ambient media. Some hyperosmoregulators show significant changes of the hemolymph osmolality when they are
confronted with increasingly dilute ambient media and usually do not succeed to invade freshwater (example: Carcinus maenas; [1660]). Other euryhaline hyperosmoregulators
stabilize their hemolymph osmolality in increasingly dilute
ambient media and succeed to survive in freshwater (example
Eriocheir sinensis). Many euryhaline hyperosmoregulators
become isosmotic in seawater or hypersaline ambient media.
This group has been termed “hyperosmotic-isosmotic” regulators and is distinguished from “hyperosmotic-hypoosmotic”
regulators (1087). Species of the latter group of Crustacea
behave like hyperosmoregulators in dilute environments, but
maintain lower osmotic concentrations in the body fluids
when migrating to seawater or hypersaline waters. Probably
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Comprehensive Physiology
Osmoregulation and Excretion
the most prominent example of these crustaceans is the brine
shrimp, Artemia salina, maintaining a remarkably hypoosmotic hemolymph up to external salinities of 30% NaCl (360).
Intracellular Osmoregulation
Changes of hemolymph osmolality can be very large in crustaceans that migrate between ambient media of different
osmotic strength. Such changes have an evasive nature. If
hyperosmoregulators reduce the osmotic gradient across the
body surface, the passive loss of salt and the passive influx
of water are reduced, decreasing the burden on the energyconsuming, compensatory mechanisms (active salt absorption and urine production; see below). In this regard it is not
surprising that freshwater crustaceans usually show the lowest hemolymph osmolalities. On the other hand, the sometimes dramatic changes of hemolymph osmolalities observed
in animals that migrate between ambient media of different
osmotic strength pose the question of how the cells respond
to this challenge. Two characteristics of animal cell membranes are important in this regard: significant water permeability and the inability to withstand pressure gradients.
Consequently, intracellular and extracellular osmolality must
be matched to avoid cell damage. The regulation of the intracellular osmotic strength in response to extracellular changes
is called intracellular osmoregulation or cell volume regulation. Even in isosmotic extracellular medium, animal cells
must actively maintain their volume by using the Na+ /K+ ATPase to create a situation called double-Donnan or pump
and leak (cf. [1124]). Because of the high water permeability of cell membranes, the cell volume rapidly changes when
cells are exposed to an anisoosmotic extracellular medium.
Two basic strategies are known to maintain the cell volume
in such a situation. As a very rapid response to cell volume
changes, animal cells transport inorganic ions into or out of
the cell, and water follows osmotically, adjusting and maintaining the cell volume. This rapid mechanism of cell volume
regulation, termed regulatory volume increase (RVI) or regulatory volume decrease (RVD), depending on a hypoosmotic
or a hyperosmotic challenge, was mainly studied in isolated
cells of vertebrates (for a recent review, see [783]); similar
investigations with crustacean cells have not been conducted.
The second basic strategy of intracellular osmoregulation or
cell volume regulation is based on a response of the pool of
organic osmolytes to a change of the extracellular osmolality.
A very informative study of the response of the intracellular
amino acid pool was conducted with Chinese crabs (Eriocheir
sinensis). The results are summarized in Fig. 5 (from [611]).
Chinese crabs adapted to seawater were transferred to
freshwater, and the water content and amino acid concentration of muscle cells was monitored. The tissue water content
showed a rapid peak before it stabilized after about one day.
Concomitantly, the amino acid content of the muscle tissue
dropped, indicating that the reduction of intracellular amino
acids stabilized the cell volume. At the end of the experiment,
Water content
(%)
Amino nitrogen (γ alanine / kg WW)
80.0
300
77.5
250
Blood : 594 ± 18
Muscle : 586 ±12
75.0
Blood : 1047 ± 24
Muscle : 1022 ± 36
72.5
200
70.0
0 1
4
9
15
Acclimation time (days)
Figure 5 Volume regulation in muscle cells of Chinese crabs (Eriocheir sinensis). Animals acclimated to seawater were transferred to freshwater, and the tissue water content
(open circles) and the amino acid concentration (open squares) were monitored over
time. Hemolymph and cell osmolality amounted to approximately 1,000 mosmol kg−1
in seawater and dropped to approximately 590 mosmol kg−1 in freshwater. A different
group of animals was acclimated to freshwater and then transferred to seawater, and the
tissue water content (closed circles) and the amino acid concentration (closed squares)
were again monitored. Redrawn after (611), with permission of John Wiley and Sons Ltd.
Volume 4, April 2014
419
Osmoregulation and Excretion
hemolymph and muscle tissue reached a new osmotic equilibrium at approximately 600 mosmol/kg. When the experiment
was reversed, transferring freshwater-adapted crabs to seawater, a transient drop of the tissue water content was observed.
During the phase of recovery, amino acids in muscle tissue
were increased until a new equilibrium was reached with an
osmolality of approximately 1,000 mosmol/kg in hemolymph
and muscle tissue. Similar studies confirmed the role of amino
acids in intracellular volume regulation in isolated tissue
and in other crustacean species. Apart from the amino acids
involved (taurine and some quaternary ammonium derivatives
are also of some importance), the significance of transport of
amino acids into or out of cells, of storage/liberation of amino
acids in/from protein, as well as of their synthesis/breakdown
has been addressed in (611-613, 1635). Whether cell volume
regulation based on the transport of inorganic ions (see RVD
and RVI above) is of importance in Crustacea to stabilize the
cell volume during the first phase after an osmotic challenge
remains to be addressed in the future.
Body Surface Permeabilities
Another evasive strategy of osmotic regulation is the reduction of the permeability of the body surface for passive flows
of salt and water. Apart from studying hemolymph osmolalities and ion concentrations, whole animals were also used to
determine whole body surface permeability, cf. (941, 1085).
Not surprisingly, osmoconforming crustaceans show the highest body surface permeabilities for water and salt. Euryhaline hyperosmoregulators like Carcinus maenas have significantly reduced permeabilities of their body surface (1660).
Even lower are the permeabilities of animals that succeed in
migrating to freshwater (Eriocheir sinensis; [1659]), and of
animals that continuously inhabit freshwater, such as freshwater crabs (Potamon niloticus; [1658]) or crayfish (Astacus
fluviatilis; [215]). Also hypoosmoregulating crustaceans like
Artemia salina (1717) or fiddler crabs (65) were found to have
significantly reduced water permeabilities of the body surface
(cf. [941]). Changes of body surface permeabilities seem to
appear over a time-course of days. However, the structural
and mechanistic changes involved are poorly understood and
require further investigation.
Water balance
Reduced osmotic gradients and body surface permeabilities
are evasive strategies of osmotic regulation. The compensatory mechanisms—active salt absorption and urine production in hyperosmoregulators, and active salt secretion and
drinking in hypoosmoregulators—have also been studied on
whole animals.
The first compensatory mechanism in osmotic regulation
discussed here is the adjustment of water volumes. For hyperosmoregulators, a passive gain of water has to be compensated for by excretion of urine. Crustaceans use their antennal glands, also called green or renal glands, or the maxillary
420
Comprehensive Physiology
glands to produce urine. It should be noted that crustaceans are
ammoniotelic. Instead of the antennal and maxillary glands,
the gills are the primary site of excretion of nitrogenous waste
(cf. the section below on Excretion). Anatomically, the crustacean antennal glands consist of a coelomic end sac, an excretory tubule, and a short duct that opens to the environment
either at the base of the second antenna (antennal glands) or at
the base of the second maxilla (maxillary glands; cf. [556]).
It was observed that glucose appears in crustacean urine after
injection of phlorizin (an inhibitor of glucose reabsorption),
and that inulin is concentrated (1554, 1555). From these observations it was concluded that filtration is the mechanism producing the primary urine and that the final urine is produced
by reabsorption and secretion along the tubular gland. Therefore, the antennal glands of Crustacea are anatomically and
physiologically similar to the vertebrate nephron (1549). It
has been shown that the urine volume responds significantly
in weak hyperosmoregulators to the increased passive water
influx in dilute ambient medium. For example, osmoconforming Carcinus maenas produced 4.4 ml kg−1 h−1 when in
seawater, but 21 ml kg−1 h−1 when hyperosmoregulating in
40% seawater (138). Similar results were obtained with other
euryhaline crustaceans (e.g., 1088, 1405, 1966). On the other
hand, urine flow in freshwater crustaceans can be very small
(e.g., 215, 1550, 1658). In osmoconformers and many hyperosmoregulators, the urine is isosmotic with the hemolymph
(941, 1151), and weak hyperosmoregulators lose significant
amounts of salt with the large volume of urine. On the other
hand, strong hyperosmoregulators produce much lower volumes of urine, which is certainly related to the low water
permeability of their body surface (see above). Even though
freshwater crabs produce isosmotic urine, the salt loss with the
urine is reduced because of the low volume of urine production. Some strong hyperosmoregulators, especially crayfish,
have evolved the capability of anisoosmotic reabsorption of
salt and the production of dilute final urine (1151, 1404, 1551,
1633; see below).
In seawater, the urine volume of crustaceans can be very
low and varies between 3% and 15% of the body weight per
day (cf. [1085]). For hypoosmoregulating Crustacea, some
studies indicated that the urine volume further decreases
(669), whereas in others the urine volume was lowest when
the animals were isosmotic with the external medium (1405).
Hypoosmoregulators should cover their passive water loss
by drinking like marine bony fish does (see the section on
Agnatha and Pisces below). In some hypoosmoregulating
species, drinking was demonstrated as a means of water balance (65, 371, 694, 1717).
Active NaCl transport
With regard to hyperosmoregulating Crustacea, a most
remarkable series of studies by Shaw found that the external sodium concentration for half-maximal sodium uptake
was different for different crustaceans: Carcinus maenas
(∼20 mM; [1660]), Eriocheir sinensis (∼1 mM; [1659]),
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Comprehensive Physiology
Gammarus duebeni (1.5 mM; [1661]), Astacus pallipes (0.20.3 mM; [1657, 1658]), and Gammarus pulex (0.1 mM;
[1661]). Again, these results allowed to distinguish between
different groups of hyperosmoregulators. Weak hyperosmoregulators are those animals that have a higher concentration for half-maximal saturation of salt uptake, higher body
surface permeabilities for salt and water, and higher changes
of hemolymph osmolality when exposed to different ambient media (see above). Strong hyperosmoregulators have a
low concentration for half-maximal saturation of salt uptake,
lower body surface permeabilities for salt and water, and lower
changes of hemolymph osmolality when exposed to different ambient media (see above). Studies with whole animals
also generated some insights into the mechanisms of active
NaCl absorption in hyperosmoregulating crustaceans. Krogh
(973) showed that the Chinese crab (Eriocheir sinensis) can
absorb sodium and chloride independent from each other. In
later studies that used inhibitors of transporters developed in
the meantime (479, 931, 942, 2068, 2072), further insights
were obtained with regard to the mechanisms of active NaCl
absorption in strong hyperosmoregulators (see below).
In another series of remarkable studies, Croghan (357361) generated data for the active NaCl secretion in hypoosmoregulating Artemia salina that are compatible with the
mechanisms of active NaCl secretion as they have been
described for seawater fish gills, elasmobranch rectal glands,
and the salt glands of birds and reptiles, as discussed in the
respective sections below. These studies also showed evidence for the involvement of the gills and the gut in active salt
secretion. Unfortunately, much more conclusive evidence for
active salt secretion in other crustacean species could never
be collected (see also below).
Ion and acid/base regulation
Numerous studies with whole animals focused on ion regulation in Crustacea. Calcium regulation is of particular importance for the maintenance of the exoskeleton. During intermolt, small calcium losses with the urine are probably compensated for by the calcium content of the food and/active
calcium absorption by the gills. During premolt, calcium is
absorbed from the exoskeleton and stored in gastroliths. Some
calcium is also excreted by the gills and by the hepatopancreas. During postmolt, calcium is secreted and inserted into
the new exoskeleton. During this phase, the calcium from
the stores is recovered and additional calcium is supplied by
calcium absorption from the food in the hepatopancreas and
by absorption from the ambient medium by the gills. This
scenario is based on studies with whole animals and with
biochemical and molecular approaches using tissue from the
antennal glands, the hepatopancreas, and the gills (for reviews,
see 12, 1304, 1969, 1970, 1977).
Numerous studies focused on acid/base regulation in
Crustacea (for reviews, see 241, 744, 1868, 1869, 1974).
Acid/base regulation is linked to NaCl regulation. On one
hand, it has been clearly shown that changes of ambient ion
Volume 4, April 2014
Osmoregulation and Excretion
concentrations and salinity affect acid/base regulation (735,
1867, 1869, 1971, 1981) and vice versa (1864). On the other
hand, it was shown that exchange of acid for sodium and
base for chloride can operate independent of each other in
crayfish (479).
Antennal Glands
Although transport was never studied with isolated and perfused antennal glands, measurements of osmotic and solute
concentrations along excised antennal glands allowed some
more detailed insights. As mentioned above, most Crustacea
produce isosmotic urine. However, some freshwater crustaceans evolved the capability to reduce their salt loss by
producing dilute urine. In the crayfishes Austropotamobius
pallipes pallipes and Orconectes virilis, the most dilute urine
is found in the bladder (1547, 1551, 1552). Anisoosmotic
salt absorption seems to occur in a part of the distal tubule,
which is especially long in crayfish (1436). For some crustaceans, ratios of urine and hemolymph osmolality above 1
were reported (e.g., 641). However, the only hypoosmoregulator for which significantly concentrated urine was reported
is the brine shrimp Artemia salina (cf. 1404).
Active NaCl transport in antennal glands
Apart from their evident function in the regulation of
hemolymph volume (see above), antennal glands of Crustacea
are very significant in ion regulation. The primary urine can
be considerably modified by glucose and amino acid reabsorption (84, 85, 1063, 1554), secretion of magnesium and
sulphate (262, 555, 787, 920, 933, 1063, 1089, 1256, 1404,
1602, 1625, 1633, 2066), absorption of potassium (1063,
1404, 1552) and reabsorption or secretion of calcium (9,
1063, 1404, 1568, 1972, 1975, 1976, 2067). The inulin ratio
between urine and hemolymph is usually above 1, indicating
water reabsorption following active absorption of solute, and
reflects a mechanism to reduce urine volume (1556).
The Na+ /K+ -ATPase seems to be the major ion pump
energizing transport in antennal glands. The activity of the
pump has been measured and its basolateral localization has
been verified (e.g., 921, 1436, 1607). Apical membrane of
labyrinth and bladder were used to prepare membrane vesicles and characteristics, and kinetics of specific transporters
were determined (10, 84, 85). Molecular biology techniques
allowed gene sequencing and the study of the regulation of
transporters in the antennal glands (1973).
The Gut and Hepatopancreas
The gut and hepatopancreas of crustaceans reflects a considerable surface in contact with the environment. However,
a considerable contribution of the gut and hepatopancreas
in osmoregulatory processes has never been shown (1151).
The hepatopancreas is certainly involved in the absorption
of nutrients (11, 1910), calcium regulation during the molt
421
Osmoregulation and Excretion
Comprehensive Physiology
cycle (12) and the sequestration and detoxification of heavy
metals (13).
the pleopods of isopods (1471) and for epipodites of lobsters
(1107, 1109).
Crustacean Gills
Active transbranchial NaCl absorption in strong
hyperosmoregulators
The most important organs of osmotic regulation in Crustacea
are the gills. First clear evidence for the dominant role of the
gills in active NaCl absorption was presented by Koch et al.
(953), measuring sodium absorption with isolated gills. In
the meantime it has been clearly established that the gills are
major organs of osmotic and ionic regulation in Crustacea (for
some reviews, see 556, 740, 934, 1151, 1418, 1421, 1476). In
all cases, the gills represent a significantly increased surface
area in contact with the ambient medium and protected inside
a gill chamber. Crustacean gills are multifunctional organs
involved in osmotic and ionic regulation (for references, see
above), gas exchange (1211), Ca2+ regulation (12, 1304,
1969, 1977), pH regulation (241, 744, 1868, 1869, 1974)
and excretion of nitrogeneous waste (584, 1601, 1950). The
number of paired gills, their location of attachment (pleurobranchs, arthrobranchs and podobranchs), as well as the extent
and anatomical form of surface extension (phyllobranchiate,
trichobranchiate and dendrobranchiate gills) varies between
groups and has been extensively described in Taylor and
Taylor (1811).
After the progress by Koch et al. (953), the technique of
measuring ion fluxes and transepithelial voltages with isolated and perfused gills was further developed, and active
NaCl absorption was demonstrated for a number of species
(e.g., 130, 925, 1114, 1150, 1455, 1456). For crabs, it became
evident that only the posterior gills were involved in active
NaCl absorption (1419, 1420). In many studies the transepithelial voltage was measured with isolated gills. However,
often different solutions were used to perfuse and bath the
gills, often making it impossible to analyze whether the measured voltages reflected active transport or diffusion potentials
caused by the concentration gradients and ion-selective paracellular pathways. Only later the electrogenic nature of active
NaCl absorption across crustacean gills was clearly shown
(451, 614, 1114, 1455, 1456, 1689). For active NaCl secretion in hypoosmoregulating Crustacea, the use of isolated and
perfused gills did not yet bring much further insights (1114).
Isolated and perfused crustacean gills proved very useful in
many cases, but in order to unambiguously analyze the mechanisms involved in transbranchial NaCl transport, the technique
needed to be refined. In 1989, a technique was introduced that
made use of the lamellar structure of crab gills (1640). Single lamellae were isolated and split and the resulting small
sheet of epithelium covered on the apical side with cuticle
could be mounted in small Ussing chambers. With split gill
lamellae, further methodological advancements like voltage
clamp, the use of microelectrodes (1374) and current noise
analyses (2070) became possible and were used. The electrical characteristics of the gill epithelium could be analyzed
and the mechanisms of active, transbranchial NaCl absorption
could be uncovered. Later, the same technique was used for
422
Consistent with the observations of Krogh (973) on whole
animals, active sodium and chloride absorption across the
gills of Chinese crabs (Eriocheir sinensis) can be measured
independent of each other (see Fig. 6).
When mounted in an Ussing chamber, the gill epithelium
of the Chinese crab generated a positive short-circuit current
(ISC ; reference electrode in the internal bath. A positive Isc
reflects absorption of cations or secretion of anions.) in the
absence of chloride in the external bath. This ISC was sodium
dependent and could be inhibited with internal ouabain, a
blocker of Na+ /K+ -ATPase, or with submicromolar concentrations of amiloride in the external bath. Amiloride, a blocker
of different sodium transporters, induced current-noise. Analyzing this amiloride-induced current-noise revealed the presence of epithelial sodium channels (2070). Single-channel
currents and the density of channels in the apical membrane
were determined. In the absence of sodium in the external
bath, the gill epithelium of Chinese crabs generated a negative ISC (1374, 1379). This current was chloride dependent and could be inhibited with external DIDS/SITS or
bafilomycin, or by using chloride channel blockers in the
internal medium. Inhibition of carbonic anhydrase abolished
the negative ISC , whereas ouabain did not affect it. Together,
the results indicated a mode of NaCl absorption very similar to the mechanisms described for freshwater amphibia
and fish (see Fig. 7), involving apical sodium channels, chloride/bicarbonate exchangers and V-type proton pumps, basolateral Na+ /K+ -ATPase, potassium and chloride channels,
and intracellular carbonic anhydrase that rapidly supplies protons and bicarbonate ions for the apical transporters.
With a paracellular leak conductance below 1 mS cm−2 ,
the gill epithelium of Chinese crabs is clearly a tight epithelium. Using whole animals (479, 931, 938, 942, 2068, 2072) or
split gill lamellae (1375, 1949), results were obtained, indicating that the same or a very similar mechanism of active NaCl
absorption is used by strong hyperosmoregulating Crustacea
in general.
Active transbranchial NaCl absorption in weak
hyperosmoregulators
The first studies of split gill lamellae of a weak hyperosmoregulator were conducted with Carcinus maenas and showed significant differences (1383, 1557) to strong hyperosmoregulators. Split gill lamellae generated a negative ISC that depended
on the presence of both sodium and chloride in the external
bath (see Fig. 8).
The current was inhibited with internal ouabain, barium/caesium or chloride channel blockers, and with external
caesium. This suggested a mechanism of NaCl absorption
Volume 4, April 2014
Comprehensive Physiology
Osmoregulation and Excretion
300
Gte [mS cm–2]
0
200
5
Isc [μA cm–2]
10
100
0
9
18
27
54
63
72
81
90
99
108
t [min]
–100
–200
Amiloride [μM]
Chloride
Sodium
1 10 100
0
0.01 0.1 1 10100 200
0
Independent absorption of Na+ and Cl− in the low-conductance epithelium
of posterior gills of the strong hyperosmoregulator Eriocheir sinensis. The figure shows a
representative time course of the short-circuit current (ISC ), demonstrating the negative ISC
(reflecting active, Na+ -independent Cl− absorption, further characterized in [1374]) and
the positive ISC (reflecting active, Cl− -independent Na+ -absorption, further characterized
in [2070]) across a split gill lamella. The ISC deflections are due to 10 mV voltage pulses
and are directly proportional to the transepithelial conductance (Gte ; see scale on the upper
left, redrawn after [2070]). In the presence of high external Cl− , most apical Na+ channels
are closed, and even high concentrations of external Amiloride hardly affect current and
conductance. At t = 23 min: In the absence of external Cl− (substitution with gluconate, or
at low external Cl− ; cf. [1372]), apical Na+ channels open and external amiloride reduces
current and conductance in a concentration-dependent way. At the end of the experiment,
the Na+ -dependence of current and conductance is shown by additional substitution of Na+
(TRIS). The low conductance at maximal external amiloride is a measure for the paracellular
conductance.
Figure 6
similar to the mode of NaCl absorption in the thick ascending
limb of Henle’s loop in the mammalian nephron where a Na+ 2Cl− -K+ cotransporter plays a central role (Fig. 9). Inhibitors
of the cotransporter had no effect on split gill lamellae, but
the ratio of net influx of chloride to current (approximately
2:1) and the ratio between net influxes of sodium and chloride
(approximately 1:2) were taken as considerable evidence for
the presence of such a cotransporter, which was later found
with molecular techniques (1848).
Not only the mechanism of active NaCl absorption but
also the basic epithelial characteristics were significantly different when compared to strong hyperosmoregulators. Split
gill lamellae of weak hyperosmoregulators have a high conductance (40-60 mS cm−2 ), which is dominated by the conductance of the paracellular pathway (1383, 1384, 1557).
Using different experimental approaches, this mechanism
was confirmed for other weak hyperosmoregulators (1384,
1455, 1456), including isopods (1471) and lobsters (1107,
1109). However, it seems that different species also apply
electroneutral NaCl absorption via apical sodium/proton
and chloride/bicarbonate exchangers to varying degrees
(cf. 556).
Na+
H+
CA
HCO3–
H2O
K+
CO2
Cl–
External
Figure 7
Internal
Mechanism of transbranchial NaCl absorption in strong
hyperosmoregulors, from (556). For further details, see the text.
Volume 4, April 2014
Active transbranchial NaCl absorption in land crabs
In land crabs, the gills play a somewhat unexpected role
in osmotic regulation. These animals live in humid areas
and their major osmotic problem is therefore not water loss.
Instead, the loss of salt with the isosmotic urine is more critical, especially because land crabs have high hemolymph salt
concentrations. To reduce the loss of salt, the urine is either
ingested or passed over the gills where NaCl is reabsorbed
(1254, 2019, 2020).
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Comprehensive Physiology
t [min]
0
12
24
36
48
60
72
84
Isc [μA cm–2]
–100
–200
0
–300
10
– Na+ i+o
– Cl– i+o
Gte
–2
20 [mS cm ]
30
Figure 8
Coupled absorption of NaCl in the high-conductance epithelium of
posterior gills of the weak hyperosmoregulators Carcinus maenas. The figure shows
a representative time course of the short-circuit current (ISC ), demonstrating the
effects of either Na+ substitution (choline) or Cl− substitution (gluconate) on both
sides of a split gill lamella mounted in a modified Ussing chamber. Current deflections are due to 5 mV voltage pulses and are directly proportional to the transepithelial conductance (Gte ; see scale for Gte in lower right, redrawn after [1383]).
The larger Gte decrease in the absence of Na+ indicates that the paracellular conductance is selective for Na+ .
Active transbranchial NaCl secretion
In hypoosmoregulating Crustacea, salt must be actively
secreted. Results obtained with hyporegulating fiddler crabs
indicated that active NaCl secretion takes an extra-renal pathway (65, 510, 641). In Artemia salina, active NaCl secretion
was related to the gills (358), and ultrastructural analyses
supported the involvement of the gills for other crustaceans
as well (1113, 1170). Isolated gills of hyporegulating Neohelice (Chasmagnathus) granulata generated a large net efflux
+
–
Na+
K+
Na+
2 Cl–
K+
Cl–
–
HCO3
H+
CA
CO2
H2O
Na+
K+
External
Figure 9
Internal
Mechanism of transbranchial NaCl absorption in weak
hyperosmoregulators (from [556]). For further details see the text.
424
of sodium when the gills were perfused and bathed with identical salines (1114), and this active secretion depended on a
functioning Na+ /K+ -ATPase. However, the complete mechanism of active, transbranchial NaCl secretion in hyporegulating crabs is unknown. Interestingly, the transepithelial voltage
was outside positive, which is opposite to the expectation for a
mechanism similar or identical to hyporegulating marine fish.
Future studies must show whether Crustacea evolved mechanisms of active NaCl secretion different from those of the
vertebrates.
Many transporters involved in transbranchial ion transport, including ion pumps, cotransporters, exchangers, ion
channels and the carbonic anhydrase, have been identified,
characterized and localized with various techniques, including pharmacology, biochemistry, histochemistry, immunohistochemistry and molecular biology (cf. 556, 1110, 1151, 1418,
1421, 1846, 1848). The influence of the gill cuticle on transbranchial ion transport (reviewed in [1050]) relates to an additional permeability barrier and the generation of an additional
compartment, the subcuticular space. Although the electrical
resistance of gill cuticles of actively transporting gills is low
when compared to the resistance of the epithelium, ion selectivity and rectification that were observed in a number of cases
(56, 1051) could interfere with the overall transport process.
More detailed studies are required in the future to elucidate
the role of the gill cuticle. In any case, it is worthwhile to
underline that certain drugs have been shown to interfere with
the cuticle (1380), which may result in misinterpretations in
pharmacological studies.
Some anatomical and functional specializations in the
gills of crabs and crayfish have been discussed in a recent
review (556).
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Comprehensive Physiology
Modulation of Osmotic and Ionic Regulation by
Hormonal and Non-Hormonal Factors Apart from
studying the changing capabilities and mechanisms of
osmotic regulation during the ontogenetic development of
crustaceans (for reviews, see 46, 272, 273), almost all studies of the hormonal and non-hormonal osmotic regulation in
crustaceans have been performed with adult animals of euryhaline species. These animals migrate between different salinities and have the capability to acclimate to changes in ambient
osmolalities. In some species, such migrations between waters
of different salinities are essential prerequisites for a successful larval development. Many studies monitored changes of
hemolymph osmolality and ion composition in response to
changing ambient osmolalities (cf. 1151; see also above and
Fig. 4). Most studies focused on the most prominent organ of
osmotic regulation in Crustacea: the gills.
No signs of active NaCl absorption could be detected in
osmoconform crustaceans from seawater. In contrast, active
NaCl absorption was measured as net influxes of sodium
and/or chloride, transepithelial electrical voltages or currents
after the animals were acclimated to dilute ambient media
(e.g., 1108, 1372, 1373, 1418, 1421). In the Chinese crab
Eriocheir sinensis, the paracellular conductance was observed
to be significantly decreased after acclimatization to dilute
ambient media (1372).
Comparing osmoconforming and hyperosmoregulating
animals (hyperosmotic-isosmotic regulators) of the same
species showed sometimes dramatic changes in gill structure
(e.g., 331, 339, 547, 1170, 1214, 1215, 1421). Whereas the
gill epithelial cells are thin in osmoconforming animals, the
gill epithelium of hyperosmoregulating crustaceans is thick
and the cells display extensive systems of apical and basal
infoldings. The number and density of mitochondria is usually increased in euryhaline hyperosmoregulators, reflecting
a higher energy requirement in these animals. In palaemonid
shrimps, significant structural changes can be observed in the
epithelial pillar cells and the adjacent septal cells. In some
hyperosmotic-hypoosmotic regulators, the structural features
typical for hyperosmoregulation do not disappear when the
animals are acclimated to seawater or hypersaline ambient
media where these animals behave as hyporegulators (533,
1113, 1114, 1170).
Concomitantly with the structural changes, increases in
Na+ /K+ -ATPase activity (e.g., 1152, 1153, 1422, 1687, 1741,
1931; reviewed in 1110, 1151, 1418, 1421, 1845) and mRNA
expression (845, 1101, 1110, 1115, 1848) have been observed
in a number of hyperosmoregulating species. Similarly, augmented carbonic anhydrase activity and mRNA expression
has been found (e.g., 156, 157, 731-739, 741-743, 1370,
1460, 1649, 1650). mRNA expression of V-ATPases was
increased in the strong hyperosmoregulator Eriocheir sinensis (1955). Interestingly, in the hyper-hyporegulator Neohelice
(Chasmagnathus) granulata, mRNA expression of Na+ /K+ ATPase and Na+ -2Cl− -K+ cotransporters was dramatically
increased in the posterior gills of hyper- and hyporegulating
crabs when compared with the gills of isosmotic crabs (1115).
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Osmoregulation and Excretion
This finding indicates the involvement of these transporters
in active NaCl absorption when the animals are exposed to a
dilute environment and in active NaCl secretion when the animals are hyporegulating in hypersaline ambient medium. In
crabs, the structural and functional changes reviewed above
are limited to the posterior gill pairs. The anterior gills of
crabs do not respond to salinity changes and have apparently
no role in osmotic regulation.
In strong hyperosmoregulating Chinese crabs acclimated
to freshwater, changes of the hemolymph-side osmolality resulted in alterations of the rates of active absorption of sodium and chloride measured as short-circuit currents (1371). Because increasing the osmolality resulted in
a decrease of NaCl absorption and decreasing the osmolality increased active transport, the observation was termed
autoregulation. It was assumed that the transport modulation is based on cell volume changes, because the addition
of membrane-impermeant sucrose reduced transport rates,
whereas the addition of membrane-permeant urea increased
them. Another observation with importance for regulatory
mechanisms was an interaction between transcellular chloride and sodium absorption (1372). In the presence of low
external NaCl, sodium and chloride absorption short-circuit
each other. At higher external NaCl concentrations, transcellular chloride absorption can be monitored, whereas transcellular sodium absorption is abolished or significantly reduced.
Transcellular sodium absorption becomes visible only when
chloride absorption is inhibited. It appears that higher rates
of chloride absorption induce an intracellular change of so
far unknown nature that inhibits transcellular sodium transport. In vivo, NaCl absorption could be rapidly adjusted with
the underlying mechanism by modulating the apical sodium
permeability and by directing sodium fluxes across the apical
membrane (high transport rates in very dilute ambient media)
or across the low conductance paracellular junctions (low
transport rates at increased ambient NaCl). After acclimation
of the animals to brackish waters (9% or 18% salinity) the
paracellular conductance was still below 1 mS/cm2 , whereas
it significantly increased after acclimation to full-strength
seawater. Autoregulation by the hemolymph-side osmolality
was also observed in the weak hyperosmoregulator Neohelice (Chasmagnathus) granulata (1860). The results of the
latter study indicated that the stimulating effect of reduced
hemolymph-side osmolality was based on a Na+ /K+ -ATPase
activation and mediated in part by an increased intracellular cAMP concentration. Activation of adenylate cyclase by
mechanical stretch of the membrane is indeed known from
vertebrates (1935).
Increased levels of cAMP were also found to stimulate
transport rates in the strong hyperosmoregulators Eriocheir
sinensis (1558). Sodium absorption was shown to be stimulated by increasing the number of open sodium channels
in the apical membrane, whereas the cAMP-induced stimulation of chloride absorption was accompanied by a significant increase of the electromotive force, suggesting a
cAMP-induced increase in V-ATPase activity. Subsequently,
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Osmoregulation and Excretion
Comprehensive Physiology
an extract of the eyestalks was shown to mimic these effects
(1382), and it was proposed that one of the peptide hormones from the sinus gland may be involved in the regulation
of active, transbranchial NaCl absorption in Chinese crabs.
Dopamine also induced an increase in intracellular cAMP
in the gills of Chinese crabs, stimulating sodium influxes
by increasing Na+ /K+ -ATPase activity (1241, 1242). Also,
in weak hyperosmoregulators, dopamine was observed to
increase intracellular cAMP in the gills and to stimulate active
salt absorption via activation of Na+ /K+ -pumps (594, 689,
880, 881, 1730). In the weak hyperosmoregulator Pachygrapsus marmoratus, crustacean hyperglycemic hormone (CHH)
was shown to stimulate salt absorption (1739), and in freshwater crayfish, CHH injection prevented salt loss after eyestalk
removal (1648).
Despite the wealth of data about osmotic and ionic regulation in Crustacea, further, more detailed analyses are necessary for a more complete understanding of the mechanisms
of osmotic and ionic regulation and their modulation by hormonal and non-hormonal factors.
Insecta
Design of Insect Osmoregulatory and
Excretory systems
The excretory system of insects is comprised of the
Malpighian tubules and the gut, especially the hindgut
(Fig. 10). Each Malpighian tubule consists of a single layer
of squamous epithelial cells that form a blind-ended tube.
Na+,K+,
Cl–
H2O
Tubules range from 2 mm to 70 mm in length, 2 to 250 in
number and up to 100 μm in diameter (1438).
Insects provide a study in contrasts with the vertebrates
in terms of the sites and mechanisms of urine formation. In
most vertebrates, blood pressure is the driving force for ultrafiltration across the glomerulus of the kidney. The resulting
primary urine is modified by reabsorption of salts and water
in the loop of Henle and the collecting ducts. The exoskeleton
precludes the development of significant hemolymph pressure in most insects, so ultrafiltration is not feasible. Instead,
formation of the primary urine involves active transepithelial
secretion of ions (primarily K+ , Na+ and Cl− ) into the lumen
of the Malpighian tubule and passive flow of osmotically
obliged water. The primary urine is modified subsequently by
reabsorptive or secretory processes in the hindgut, although
in a few species such modification occurs in a downstream
segment of the tubule itself. The higher ratio of surface area
to volume for tubule cells to tubule lumen, relative to hindgut
cells to hindgut lumen, presumably allows for more rapid processing of the urine in insects subject to fluid loading, such
as blood-feeding hemipterans and the fruit fly Drosophila
melanogaster.
The bulk of water, ion and metabolite reabsorption occurs
in the rectum, resulting in strongly hyperosmotic or hypoosmotic excreta. In many terrestrial species of insect, the hindgut
can recover virtually all the water from the gut contents and
fluid secreted into the gut by the Malpighian tubules. The
resulting fecal pellets are powder-dry in species such as the
mealworm Tenebrio molitor (1503) and the firebrat Thermobia domestica (1324). High concentrations of solutes, wastes
Isosmotic
secretion
Most water,
ion and
metabolite
reabsorption
Some KCI
and water
reabsorption
Midgut
Rectum
Anterior
hindgut
Malpighian tubule
Diffusion of
small solutes
Hypoosmotic or
hyperosmotic
excreta
Active
secretion of
toxins (OAs,
OCs)
Figure 10
Schematic diagram of the insect excretory system. Active transport is indicated
by solid arrows and passive transport is indicated by open arrows. The flows of the gut
contents and the fluid secreted by the Malpighian tubules are indicated by dashed arrows.
Adapted from Phillips (1438), with modifications. Abbreviations: organic anions: OAs;
organic cations: OCs.
426
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Comprehensive Physiology
and toxins are produced in the lumen as a consequence of
water recovery (1443). The cuticular lining of the hindgut
thus plays an important role in protecting the transporting
cells of the rectal epithelium from lumen contents.
Processes such as desiccation, feeding or osmotic influx
across the external surfaces of aquatic species create the
need for adjustments of hemolymph fluid and solute levels.
However, several features of insect physiology and morphology reduce the evolutionary pressure for rapid alterations
of hemolymph composition by the excretory system. First,
insects do not depend on a blood-borne respiratory pigment
whose effectiveness would be altered by changes in the volume and composition of the extracellular fluids. Instead, respiratory gas exchange is accomplished by the ramifying tubes
of the tracheal system whose termini extend into cells and
tissues. Second, an effective blood-brain barrier protects the
eyes and central nervous system from extracellular toxins
or changes in hemolymph ionic composition. Epithelia such
as the perineurium, which envelopes the insect central nervous system, are characterized by the presence of effective
cell-cell junctions and by inorganic ion and xenobiotic transporters that protect the neuronal tissues (1188, 1857). Third,
the waxy cuticle limits water loss, so that terrestrial insects are
protected from rapid desiccation in spite of their large surface
area-to-volume ratio.
Given the coevolution of insects and flowering plants and
the large number of plant-derived toxins that have evolved to
minimize herbivory, a key role of the insect excretory system
is to automatically eliminate novel toxins. The Malpighian
tubule provides a means for removal from the hemolymph
of all soluble substances of low molecular weight, and in this
respect it provides analogies with both the glomerulus and the
tubule of the vertebrate nephron. It is somewhat unexpected,
therefore, that the absolute permeability of the Malpighian
tubule epithelium of insects is quite low when compared to
the excretory epithelia of other invertebrates and the vertebrate kidney. Given the need to clear toxic compounds from
the hemolymph, this characteristic seems paradoxical (1346,
1706). However, it is the area for passive permeation, namely
the paracellular clefts, that is restricted, rather than the permeability of these sites. Adjacent cells in insect epithelia, such
as the Malpighian tubule, are separated by septate junctions
rather than the occluding, or tight, junctions of transporting
epithelia in vertebrates (988). Although the ratio of the areas of
the basal side of the cells (i.e., the side facing the hemolymph)
and the intercellular cleft is 3,000:1, the ratio of transcellular to paracellular area increases to 120,000:1 because of the
40-fold amplification of basal surface area created by membrane folding and convolution (1706).
The rates of passive diffusion of solutes across the tubule
epithelium vary inversely with molecular mass (372, 1135).
Uncharged solutes that have a molecular mass greater than
that of sucrose, as well as charged solutes, are excluded from
the tubule cells and can cross only via the intercellular junctions. Uncharged solutes whose molecular mass is less than
that of sucrose, including relatively large molecules such as
Volume 4, April 2014
Osmoregulation and Excretion
mannitol, cross into the lumen of the upper Malpighian tubule
of Rhodnius by a cellular route (1346). Rapid permeation by
the transcellular route in Rhodnius is a consequence of the
large area of cell membrane available.
Whereas the vertebrate glomerulus passes compounds
as large as inulin, whose molecular weight is ∼ 5,000, the
Malpighian tubules of insects secrete fluid that contains even
such small molecules as sugars and amino acids at concentrations much below those in the hemolymph. Toxic molecules,
even those of moderate size, can diffuse through the intracellular clefts and are removed passively, albeit slowly, from the
insect’s hemolymph (1127). One advantage of this arrangement is that less energy is required to produce the primary
excretory fluid and to reabsorb useful substances from it.
Also, high concentrations of molecules such as amino acids,
trehalose and lipids can be maintained in circulation. Trehalose concentrations may exceed 100 mM in the hemolymph
of flying insects, where the sugar is used as fuel for the flight
muscles (1962). Many species of butterflies, ants and beetles
contain levels of amino acids in the hemolymph as high as
100-200 mM (1128). In addition, hormones that are released
into circulation will also be removed slowly by the excretory
system, and the amounts that must be released may therefore
be reduced.
Insects can eliminate excess fluid at very high rates during diuresis, yet they lose only trace amounts of hemolymph
solutes. Given that many hemolymph solutes of even quite
small molecular size only appear in the filtered fluid at concentrations in the 10%-50% range of those in the insect’s
hemolymph, reabsorption of such solutes need only occur in
insects at about 1%-2% of the rate required in many vertebrates, which filter their extracellular fluid 10-20 times more
rapidly than do most insects. There is thus a large saving in the
energetic costs associated with reabsorption of solutes from
tubule lumen (1127).
In addition to passive diffusion of solutes from
hemolymph to tubule lumen, active mechanisms rapidly transport a wide range of solutes. The tubules reabsorb useful compounds, such as glucose (951), and actively secrete
organic cations, organic anions, urate, phosphate, magnesium, sulphate, sulphonates, acylamides, alkaloids and glycosides (1075, 1134, 1334, 1536, 1588, 1844). Studies using
tubule-specific DNA microarrays reveal that the Malpighian
tubule is an epithelium that is richly endowed with solute
transporters, including transporters for many potential toxins
(447). The comprehensive and paradigm-shifting research of
Dow, Davies and coworkers (285, 446, 447) has documented
the enrichment of solute transporters in the tubule genome
and suggests a reinterpretation of the primary physiological
role of the Malpighian tubules. Rather than just regulating
inorganic ion levels in the hemolymph and whole animal
water balance—tasks that may be done downstream in the
hindgut—the primary role of the tubules may be the regulation of organic solute levels in the hemolymph. Indeed, Dow
and Davies (447) point out that in view of electrophysiological data indicating that the tubule is analagous to a vertebrate
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Osmoregulation and Excretion
tight epithelium (125), its apparent leakiness may stem from
high levels of transcellular organic solute transport, rather
than paracellular diffusion.
Lastly, the insect excretory system shows complex interactions between the rates of inorganic ion transport, fluid secretion and the elimination of toxins. In particular, a high degree
of phenotypic plasticity is seen when insects are exposed to
dietary toxins. When Drosophila, for example, are reared on
diets enriched in organic anions such as salicylate or fluorescein for one day, there is an increase in fluid secretion rate
of up to threefold and an increase in organic anion transport
of up to fivefold. Since fluid secretion is a passive osmotic
consequence of the transport of K+ , Na+ and Cl− , these findings indicate that dietary toxin exposure leads to an upregulation of the expression and/or activity of transporters for
both organic anions and for inorganic ions (265, 1590). Similarly, when insects are exposed to dietary or environmental
salt stress, there is an increase in the capacity to secrete the ion
present in excess (443, 1287). For Drosophila, the increase in
K+ secretion in response to K+ -rich diets is also associated
with a 60% increase in the fluid secretion rate (1288). Earlier
studies have shown that the capacity to eliminate uric acid,
organic anions or Ca2+ increases in tubules isolated from the
blood-feeder Rhodnius at intervals after the blood meal corresponds to the appearance of these ions in the hemolymph
(1130, 1139, 1334). It thus appears to be a general finding
that insect Malpighian tubules show pronounced phenotypic
plasticity in response to dietary loading with toxins or ions.
The epithelium is effectively remodeled so that its capacity to
secrete compounds present in excess is increased. Presumably
this remodeling is advantageous in that there is not the high
metabolic cost of maintenance of transporters when they are
not required.
Secretion of Physiological Ions by Insect
Malpighian Tubules
Malpighian tubules composed of a single cell type
Much of our knowledge of tubules composed of a single
cell type comes from studies of the blood-feeding hemipteran
Rhodnius prolixus, the ant Formica polyctena and the omnivorous orthopteran Acheta domesticus. When stimulated with
diuretic factors, the Malpighian tubules of Rhodnius secrete
fluid at prodigious rates, equivalent to each cell secreting a
volume of fluid equal to its own volume every 15 seconds
and an amount of Cl− equivalent to the cellular Cl− content
every 3 seconds (1140, 1340). Together, the four Malpighian
tubules eliminate urine at a rate equivalent to the insect’s
unfed body weight every 20 to 30 minutes. The large diameter (90 μm) and length (30 mm) of the secretory segment
facilitates setting the tubules up in the in vitro Ramsay assay
(Fig. 11), where they secrete fluid at ∼70% of the in vivo
rate. The large cells (∼ 100 μm) in Rhodnius tubules facilitate impalement with double-barrelled ion-selective microelectrodes. Such measurements allow the electrochemical
428
Comprehensive Physiology
Data acquisition
system
Metal pin
Reference
microelectrode
Ion-selective
microelectrode
Measure
[Ion],
radioactivity,
fluorochrome
intensity
Paraffin oil
Sylgard
Saline
Tubule
Secreted fluid
Figure 11
Schematic of the Ramsay assay for measurement of
Malpighian tubule fluid secretion rate and secreted fluid solute concentration. An isolated Malpighian tubule is placed in a drop of saline
under paraffin oil, and the open end of the tubule is secured to a pin
embedded in the Sylgard-lined bottom of the petri dish. Secreted fluid
collects at a puncture in the wall of the tubule between the bathing
saline and the pin. Secreted fluid ion concentration ([Ion]) is measured
using ion-selective microelectrodes (1288), atomic absorption spectroscopy (923) or energy-dispersive X-ray microanalysis (1902, 1998).
Organic solute concentrations can be determined using liquid scintillation spectrometry of radiolabeled solutes (1536) or by confocal
laser-scanning microscopic measurement of fluorescence intensity of
droplets collected in optically flat glass capillaries (1023). Solute secretion rate (flux) is typically in the femtomol min−1 to picomol min−1
range and is calculated as the product of fluid secretion rate (nl min−1 )
and solute concentration (μmol l−1 to mmol l−1 ).
gradients for Na+ , K+ , Cl− and H+ across the basolateral
and apical membranes to be measured. In conjunction with
measurements of ion activities in the tubule lumen, these data
are essential for evaluating the thermodynamic feasibility of
specific channels and other membrane transporters such as
Na+ /H+ exchangers and Na+ -2Cl− -K+ cotransporters (821,
822, 824).
Rhodnius feeds on blood that is approximately
14% hypoosmotic to its hemolymph osmolality of 370
mosmol kg−1 (1133). Homeostasis is maintained by producing hypoosmotic urine through a two-stage process: Na+ , K+ ,
Cl− and water are secreted across the upper Malpighian tubule
and K+ and Cl− but not water are reabsorbed downstream
in the lower Malpighian tubule, as described in a subsequent
section.
The upper segment of the Malpighian tubules of Rhodnius prolixus secretes fluid containing approximately 100 mM
NaCl and 80 mM KCl during diuresis (1133). Secretion of
ions and osmotically obliged water by tubules of Rhodnius and
other species is driven primarily by an apical vacuolar-type
H+ -ATPase (1137, 1992). Aquaporins are present in both the
upper secretory tubule and the lower Malpighian tubule (471),
and expression increases in response to blood feeding or treatment of isolated tubules with serotonin or cAMP (1178).
Current models propose that all ion transport is transcellular (578, 821, 824). Paracellular transport of Cl− , as proposed
for mosquito tubules composed of two cell types (see below),
is precluded by the lumen-negative potential in Rhodnius.
It is suggested that electrogenic transport of H+ from the
cell to the lumen energizes amiloride-sensitive exchange of
Volume 4, April 2014
Comprehensive Physiology
Osmoregulation and Excretion
(A)
H+
ATP ADP+Pi
Na+,
K+
Cl–
H+
Na+ K+ Cl– Cl–
cAMP
5HT
(B)
H+
Na+,
K+
Cl–
ATP ADP+Pi H+
Na+ K+ Cl– Cl–
Ca2+
ATP
Achdo-KII
K+
ADP+Pi
Na+
Figure 12
Schematic of ion transport processes involved in fluid
secretion by (A) 5HT-stimulated upper tubules of Rhodnius (822, 1342)
and (B) Achdo-KII-stimulated principal cells in the main segment of
cricket Malpighian tubules (316). Dotted lines are used to illustrate
the intracellular signaling pathway. Solid circles represent primary
active transport processes, whereas open circles are used to represent cotransporters and antiporters. The dark blue symbols represent
the receptors for 5HT and for Achdo-KII and their associated ligands.
cytoplasmic K+ and/or Na+ for luminal H+ . The primary
route of entry of Na+ , K+ and Cl− is through a bumetanidesensitive basolateral Na+ -2Cl− -K+ cotransporter (Fig. 12A).
A characteristic triphasic change in transepithelial electrical potential (TEP) has been reported when fluid secretion
by isolated Malpighian tubules of Rhodnius prolixus is stimulated by serotonin (1345). From the initially negative value in
unstimulated tubules (−25 mV, lumen-negative), TEP shifts
to −33 mV in phase 1, −30 mV in phase 2 and −32 mV in
phase 3. The results of ion substitution and addition of specific pharmacological reagents suggest that the three phases
of the response of TEP to serotonin correspond to sequential activation of an apical Cl− channel, an apical V-type
H+ -ATPase and a basolateral Na+ -2Cl− -K+ cotransporter.
Bathing tubules in Cl− -free saline before and during 5-HT
stimulation abolishes phase 1, whereas preincubation in the
presence of the H+ -ATPase inhibitor bafilomycin abolishes
phase 2. Bathing tubules in saline containing bumetanide,
Volume 4, April 2014
an inhibitor of Na+ -2Cl− -K+ cotransporters, before stimulation abolishes phase 3 but does not alter phases 1 and 2.
An increase in apical Cl− conductance during phase 1 may
enhance movement of Cl− across the apical membrane from
cell to lumen, producing the negative going shifts in TEP
during this phase. The transepithelial potential reflects contributions of the V-type H+ -ATPase, tending to drive the lumen
to more positive values, and compensating movement of Cl−
from cell to lumen, tending to drive the lumen to more negative values. After apical permeability to Cl− increases in
phase 1, the apical membrane potential is strongly influenced
by changes in intracellular Cl− activity following enhanced
entry of Cl− into the cell by activation of a basolateral Na+ 2Cl− -K+ cotransporter in phase 3. This cotransporter is the
dominant mechanism of Cl− entry. Removal of Na+ from the
bathing saline or addition of bumetanide thus has the same
effect on apical membrane potential as does exposure to Cl−
free saline; the lumen rises to a dramatically positive potential, because the H+ -ATPase activity is not compensated by
entry of a counterion (825, 1345). Na+ -2Cl− -K+ cotransport has also been implicated in basolateral entry of ions
into Malpighian tubules of most insect species that have been
studied (1337).
As noted by Coast (316), the mechanism of ion transport
across the secretory tubules of Rhodnius is strikingly similar to that seen in tubules of the cricket Acheta domesticus
(Fig. 12B). Cricket tubules also lack stellate cells and Cl− is
transported across the principal cells and into the lumen via a
basolateral Na+ -2Cl− -K+ cotransporter and apical Cl− channels. In unstimulated tubules, much of the Na+ that enters
cells via the cotransporter is returned to the bathing medium
by a basolateral Na+ /K+ -ATPase (1138), so the secreted fluid
is K+ -rich. When the tubules are stimulated with serotonin
(Rhodnius) or Achdo-DH (A. domesticus), the increase in
Na+ -2Cl− -K+ cotransporter activity (321, 825, 1345) overwhelms the activity of the Na+ /K+ -ATPase and the additional
Na+ is exported to the lumen (1138). The Na+ /K+ ratio of
the secreted fluid thus increases from 0.62 to 1.35 in Rhodnius (824) and from 0.23 to 1 in A. domesticus (316). The
higher Na+ /K+ ratio for tubules of Rhodnius may indicate a
higher affinity of apical cation/H+ antiporters for Na+ (1132).
In spite of these close similarities in the apical and basal
membrane transporters in tubules of Rhodnius and crickets,
there are important differences. First, the transport capacities of Rhodnius and A. domesticus tubules differ enormously
when stimulated. Whereas serotonin increases secretion up
to 1,000-fold, the increase in response to Achdo-DH is 3- to
4-fold (316). Second, the effects of serotonin on Rhodnius
tubules are mediated primarily by increases in intracellular
cyclic AMP, whereas the effects of Achdo-DH on cricket
tubules are mediated, as for other kinins, through increases in
intracellular Ca2+ .
The apical vacuolar H+ -ATPase The apical vacuolartype H+ -ATPase is the primary energizer of transport in insect
epithelia, and we now have a detailed understanding of its
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Osmoregulation and Excretion
structure, function and regulation (127). V-ATPases are multisubunit, heteromeric proteins that consist of two structural
domains: a peripheral, catalytic V1 domain (∼ 500 kDa) and
a membrane-spanning V0 domain (100-250 kDa). The stator (consisting mostly of the V1 domain) anchors the pump
to the cell membrane and is responsible for ATP hydrolysis, whereas the rotor (consisting mostly of the V0 domain)
transfers H+ from one side of the membrane to the other.
Immunohistochemical evidence indicates an apical location
of the V-ATPase in insect Malpighian tubules, corresponding
to the portasomes revealed in transmission electron micrographs (1591, 2074).
An important method of regulating V-ATPase activity is
through reversible dissociation of the complex into the V1
and V0 domains. In insects, cAMP and PKA are implicated
in regulating V-ATPase activity; phosphorylation of subunit
C promotes association of V1 and V0 and consequent ATP
hydrolysis and proton translocation. Other modes of regulation include variation in the coupling ratio between ATP
hydrolysis and H+ translocation, as well as inhibition of VATPase activity by reversible formation of a disulphide bond
between two cysteine residues in subunit A of the V1 domain.
The most dramatic regulation may be indirect, through alteration of membrane Cl− permeability. Studies of another secretory epithelium, the blowfly salivary gland, provide ideas as to
how this regulation is achieved (78). V-ATPases are inherently
electrogenic because their activity pumps protons without an
associated anion. In the absence of other charge carriers, therefore, the V-ATPase generates a large voltage across a membrane and produces a modest change in H+ concentration.
When Cl− permeability is low, as in unstimulated tubule cells,
the large lumen-positive potential back-inhibits the activity of
the pump. Increased Cl− permeability (e.g., through inositol
trisphosphate-mediated increases in intracellular Ca2+ ) dissipates the electrical component of the V-ATPase activity,
thereby depolarizing the transmembrane potential and allowing pump activity to increase. Thus, when anion channels
(e.g., Cl− ) are active, the secretion tends to be more acidic.
By contrast, an alkaline secretion is generated in the presence
of a cation—nH+ antiporter or symporter and appropriate
counterions (anions).
In contrast to the Na+ /K+ -ATPase and other P-typeATPases, V-ATPases do not form a phosphorylated intermediate and are therefore relatively insensitive to vanadate,
a phosphate-mimic. V-ATPases are highly sensitive to the
potent and specific inhibitor bafilomycin A1 , a macrolide
antibiotic (452) and to archazolid (817) Fluid secretion by
the Malpighian tubules of several species examined is inhibited by 1-10 μM bafilomycin (1337).
Apical K+ /H+ and Na+ /H+ exchange The V-ATPase
creates both electrical and chemical gradients that can be
used to drive the exchange of cellular K+ (or Na+ ) for lumenal H+ across the apical membrane of insect epithelia. In
the midgut of Manduca sexta, the exchanger is electrogenic;
2H+ are exchanged for each K+ ion. Exchange is thus driven
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Comprehensive Physiology
primarily by the large lumen-positive electrical gradient established across the apical membrane by the V-ATPase (1991).
Exchange of 2H+ for each K+ or Na+ also seems to be
required in Malpighian tubules of Aedes aegypti (127), as
discussed below. By contrast, measurements of membrane
potential, intracellular and lumenal pH in Malpighian tubules
of ants (2073) and Rhodnius (824), show that electroneutral
exchange (1H+ : 1K+ or 1H+ : Na+ ) is sufficient to drive alkali
cations from cell to lumen. When Rhodnius tubules are stimulated with the diuretic factor 5-HT, intracellular pH acidifies
slightly from 6.97 to 6.92 and lumen pH shifts to a more
alkaline value, from 6.08 to 6.32 (824). Apical Na+ /H+ and
K+ /H+ exchange activity may thus be stimulated to a greater
extent than the V-ATPase, thus tending to drive the lumen
slightly alkaline and the cells slightly acid, relative to unstimulated tubules. Application of amiloride, which blocks Na+ /H+
exchangers, inhibits fluid secretion and acidifies the lumen,
consistent with continued operation of the apical V-ATPase
when apical Na+ /H+ exchange is blocked. The molecular
identity of the alkali cation:proton exchangers in the apical membrane has been the subject of several studies using
dipteran insects and is therefore discussed in more detail in
the section describing tubules with two cell types: principal
and stellate.
Na+ -2Cl− -K+ cotransport Movement of Cl− from the
hemolymph into the cell is thermodynamically uphill, thus
ruling out Cl− entry into the cells through channels (316, 821).
Bumetanide-sensitive Na+ -2Cl− -K+ cotransport is a form of
tertiary active transport of Cl− across the basolateral membrane. The V-ATPase creates the proton motive force that
energizes secretion of Na+ and K+ into the lumen through
the apical cation:proton antiporters. The reduction in cellular
Na+ thus creates a favorable gradient for Na+ entry across
the basolateral membrane. The Na+ gradient, in turn, energizes Na+ -2Cl− -K+ cotransport. Measurements with doublebarrelled ion-selective microelectrodes show that Cl− entry
via the Na+ -2Cl− -K+ cotransporter is favored thermodynamically (316, 821) (Fig. 13).
An unusual aspect of the basolateral cation:Cl− cotransporter in Rhodnius Malpighian tubules is that K+ can be
replaced by Na+ . In response to complete removal of K+
from the bathing saline, 5-HT-stimulated Malpighian tubules
secrete fluid at 45% of the rate in the control saline and remain
sensitive to bumetanide. Transport is unaltered by the drug
hydrochlorothiazide, an inhibitor of Na+ :Cl− cotransporters
in vertebrate tissues. Importantly, dose-response curves relating percentage inhibition of fluid secretion to bumetanide
concentration are identical for tubules bathed in control
(K+ -replete) and K+ -free saline, suggesting that fluid secretion involves the same bumetanide-sensitive cotransport system in the presence or absence of K+ . Kinetic analysis
also supports the idea that Na+ competes with K+ for
transport by a basolateral bumetanide-sensitive cation/Cl−
cotransporter during fluid secretion (822). Rhodnius produces copious amounts of NaCl-rich urine during diuresis,
Volume 4, April 2014
Comprehensive Physiology
pH=6.32
aK+=49
aNa+=66
aCl– =109
Osmoregulation and Excretion
ΔμH/F=–60
Lumen
ΔμNa/F=–49
Lumen
–28 mV
Cell
Cell
pH=6.82
ΔμK/F=–22
aK+=71
aNa+=33
aCl–=32
pH=7.25
aK+=6
aNa+=103
aCl – =93
ΔμH/F=–41
Bath Na+
ΔμNa/F=–95
Δμnet/F = –62 mV
–63 mV
0 mV
Bath
ΔμK/F=0
Cl–
Cl–
Cl–
ΔμCl/F=–0.4
ΔμCl/F=33
Figure 13 Mean values for electrochemical gradients, ion activities
and pH in 5-HT-stimulated Malpighian tubules of Rhodnius prolixus.
Values of pH and ion activity (aK , aCl , aNa ) were recorded 30 min after
stimulation with 1 μmol l−1 5-HT. Electrochemical gradients (μ/F, in
mV) for Na+ , K+ and Cl− across the apical and basolateral membranes
are taken from (821, 824).
essentially eliminating the nutrient-poor plasma fraction of
the blood meal and retaining the nutrient-rich blood cells.
Competition between Na+ and K+ for transport by a single bumetanide-sensitive cotransporter in the upper tubule
provides the means for reducing secreted fluid K+ concentration, thereby reducing the need for K+ reabsorption by the
lower Malpighian tubule, and at the same time enhancing Na+
elimination.
Transport of Cl− across the apical membrane Intracellular chloride activity in Rhodnius tubules declines when
the apical membrane potential becomes more lumen-positive
in Na+ -free saline, K+ -free saline or in the presence of
bumetanide (821). This decline suggests the presence of apical Cl− channels that mediate transport of Cl− from cell
to lumen when entry of Cl− into the cell across the basolateral membrane is blocked. In Na+ -replete saline, intracellular Cl− activity is near equilibrium across the apical
membrane both in unstimulated and in serotonin-stimulated
tubules, as expected if Cl− channels mediate its transport
across the apical membrane. Although fluid secretion by
Rhodnius tubules is inhibited by 4,4 -diisothiocyano-2, 2 disulphonic acid stilbene (DIDS), transepithelial potential and
intracellular pH are unaltered, as would be expected if DIDS
blocked apical Cl− channels or Cl− /HCO3 − exchangers,
respectively. Cl− channels, if present, are DIDS-insensitive,
and the effects of fluid DIDS on fluid secretion may reflect
indirect effects of DIDS, such as inhibition of mitochondrial
functioning.
Other pathways for Cl− movement, such as apical K+ :Cl−
cotransport proposed for Aedes tubules (see below) and/or
paracellular Cl− movement, can be ruled out because they
Volume 4, April 2014
Cl–
Na+ K+
Δμnet/F = –29 mV
K+
Δμnet/F = 33 mV
Figure 14 Schematic diagram showing net electrochemical potentials (μnet /F) for three cation:Cl cotransporters in serotonin-stimulated
tubules of Rhodnius prolixus. (A) Na+ -2Cl− -K+ , (B) Na+ -Cl− and (C)
K+ -Cl− . Based on data in (821).
are thermodynamically unfeasible. The calculated electrochemical gradients indicate that a K+ :Cl− cotransporter in
the apical membrane of Rhodnius Malpighian tubules would
mediate net movement of these ions from the lumen into
the cell, that is, in the opposite direction to that required for
fluid secretion (Fig. 14). Although the transepithelial potential across tubules of most insects is lumen-positive, the TEP
in fully stimulated Rhodnius tubules is lumen-negative, and
a passive paracellular pathway for Cl− movement is thus
precluded.
K+ channels Although the K+ channel blocker Ba2+
alters the basolateral membrane potential in tubules of Rhodnius or Acheta domestica, it has no effect on fluid secretion
(316). It thus appears that K+ channels are not implicated
in net transepithelial K+ and fluid secretion by Malpighian
tubules of these two species. By contrast, multiple mechanisms of K+ transport, including channels, may be necessary
in tubules of the ant, Formica polyctena because hemolymph
K+ levels are quite variable. The model of Leyssens et al.
(1044) proposes that a Na+ -2Cl− -K+ cotransporter that is
sensitive to 10−5 M bumetanide is the primary mechanism of
uptake at the lowest bathing saline K+ concentrations of 5
to 10 mM. At the intermediate concentration of 51 mM K+ ,
a K+ :Cl− cotransporter sensitive to high (10−4 M) concentrations of bumetanide becomes active. At very high bathing
saline concentrations (113 mM K+ ), K+ enters through basolateral Ba2+ -sensitive K+ channels (1044).
Basolateral K+ channels may also contribute to tubule
+
K secretion in the black field cricket Teleogryllus oceanicus (2048). As in the ant F. polyctena, hemolymph K+ and
Na+ probably vary as a result of omnivory, and K+ may
enter the Malpighian tubule cells through Ba2+ -sensitive K+
channels under some conditions. At lower hemolymph K+
concentrations, bumetanide inhibits fluid secretion by 80%
and inhibits Na+ , K+ and Cl− secretion by 60%, 70% and
50%, respectively. This result suggests that a large proportion
of net transepithelial ion transport is mediated by cotransport
of these three ions (2048).
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Osmoregulation and Excretion
Comprehensive Physiology
Malpighian tubules composed of principal
cells and stellate cells
Malpighian tubules in which the secretory segment is comprised of two structurally distinct cells known as principal
cells and secondary or stellate cells have been studied most
extensively in the mosquito Aedes aegypti and the fruit fly
Drosophila melanogaster. The model of transport that has
emerged from studies over the last decade emphasizes the
role of the stellate cells in Cl− secretion and the principal
cells in secretion of Na+ and K+ (Fig. 15). For both cations
and anions, the primary energizer of transport is the V-type
H+ -ATPase on the apical (lumen-facing) membrane of the
principal cells. The electrogenic nature of the H+ pump means
that both electrical and chemical gradients are used to drive
the transport of Na+ , K+ and Cl− . For Na+ and K+ , apical
cation:H+ exchangers of the cation:proton antiporter (CPA)
family allow lumenal H+ to leak from the lumen back into
the cell in exchange for Na+ or K+ (1535, 2046). In bacteria, related cation:proton antiporters in the Kef family are
electrogenic; for the tubules, a stoichiometry of 2H+ :1K+ or
2H+ :1K+ would allow cation secretion to be driven, in part,
by the lumen-positive potential created by the H+ -ATPase.
Microarray data and in situ analyses show that Drosophila
genes CG10806 and CG31052 are members of the CPA2
Principal cell
Stellate cell
Cl–
H+
Cl–
cGMP
NO
Ca2+
Ca2+
ATP ADP+Pi
cAMP
Kinin
Tyramine
Cl–
Na+
DH44
DH31
CAPA
(CT-like) (CRF-like)
Figure 15 Regulation of ion transporters by peptides, tyramine
and intracellular messengers in Malpighian tubules of Drosophila
melanogaster and Aedes aegypti. In Drosophila, the CAPA peptides
result in generation of cGMP through the nitric oxide pathway in
response to elevation of intracellular Ca2+ in the principal cells (908).
The calcitonin-like (CT-like) diuretic peptide DH31 and the corticotropin
releasing factor-like (CRF-like) diuretic peptide DH44 lead to increases
in intracellular cAMP levels (323, 852). Both cAMP and cGMP stimulate the electrogenic V-type H+ -ATPase, leading to an increase in
lumen-positive apical membrane potential (1341). In mosquitoes, the
CRF-like mosquito natriuretic peptide (1437) also results in enhanced
Na+ entry through a cAMP-dependent conductive pathway in the basolateral membrane of the principal cells (1611). Kinin receptors are
present in the basolateral membranes of stellate cells in tubules of
both Aedes and Drosophila (1105, 1495). Both the kinins (125, 1495,
1496, 1817) and tyramine (146-148) result in increases in intracellular Ca2+ . In Drosophila, Ca2+ is thought to stimulate an increase
in transcellular chloride permeability through actions on apical Cl−
channels. In Aedes, Ca2+ is thought to increase the permeability of the
paracellular pathway (126).
432
family and are homologues of bacterial Kef exchangers.
Immunocytochemistry localizes endogenous CG10806 and
CG31052 to the apical plasma membrane of the Malpighian
(renal) tubule (400).
Entry of Na+ and K+ into the cells across the basolateral
membrane involves multiple transporters. Na+ may exchange
for H+ through NHE3; the reaction may also contribute to
intracellular pH regulation. Bumetanide-sensitive cotransport
of Na+ -2Cl− -K+ has been shown to be important for tubules
of both Aedes (725, 1641) and Drosophila (823). In addition, a
role for the Na+ /K+ ATPase is seen in the inhibitory effects of
ouabain on fluid secretion by tubules of both mosquitoes (725)
and Drosophila (1074, 1844). Immunocytochemical evidence
has demonstrated the presence of the Na+ /K+ -ATPase in the
principal cells of the adult tubule in Aedes (1407).
Intracellular microelectrode recordings from principal
cells reveal that ouabain depolarizes the basolateral membrane of the principal cells by ∼ 8 mV from the control
value of ∼ −60 mV (1074). The Na+ /K+ -ATPase may thus
play an ancillary role in tubule secretion; its stoichiometry
of 3Na+ :2K+ means that it may enhance basolateral entry
of K+ at the expense of Na+ ; inhibition of secretion with
ouabain is associated with an increase in secreted fluid Na+
concentration of approxiimately 50% (1074), as seen previously in unstimulated tubules of Rhodnius (1138). Much
of the Na+ that enters the cell through the Na+ -2Cl− -K+
cotransporter is recycled from cell to hemolymph by the
Na+ :K+ -ATPase, contributing to secretion of K-rich fluids
(120 mM K+ : 30 mM Na+ ; [823]) by Drosophila tubules.
The contribution of the Na+ /K+ -ATPase has been underestimated because transport of ouabain by an organic aniontransporting polypeptide reduces the effective concentration
of the inhibitor (1844). This had lead to the erroneous conclusion that the Na+ /K+ ATPase was not present in insect
Malpighian tubules. The Na+ /K+ -ATPase can be viewed as
an ancillary transporter whose roles are most apparent in
tubules secreting at the basal rate. Under such conditions, it
can play an important role in setting the Na+ :K+ ratio of the
secreted fluid and in maintaining the Na+ gradient across the
basolateral membrane for Na+ -coupled transporters such as
those implicated in elimination of small hydrophilic organic
anions. When tubules respond to diuretic factors, the apical H+ -ATPase is stimulated and plays a dominant role in
transepithelial secretion.
Chloride ions that enter through the Na+ -2Cl− -K+
cotransporter may exit the cell into the lumen through
apical Cl− channels. These may be in the principal cells,
as is the case for insects with only a single cell type in
the tubules, or Cl− may exit through Cl− channels in the
apical membrane of the stellate cells (1329, 1347). Although
dense accumulations of Cl− channels have been identified
in stellate cells of Aedes tubules, the latter pathway would
require gap junctions between the principal cells and stellate
cells (126). Alternatively, transepithelial secretion of Cl−
may involve basolateral entry into the principal cells through
a Cl− /HCO3 − exchanger (1453).
Volume 4, April 2014
Comprehensive Physiology
Measurements with double-barrelled ion-selective microelectrodes reveal that the electrochemical potential favors passive movement of K+ from cell to bath for the principal cells
of Drosophila melanogaster (823). A direct contribution of
K+ channels to transepithelial fluid secretion can thus be ruled
out. Similar results were reported in unstimulated Malpighian
tubules of Rhodnius prolixus (821). K+ channels may play a
role in transepithelial ion transport in tubules of other species
if intracellular K+ activity is below electrochemical equilibrium across the basolateral membrane, as proposed for
Malpighian tubule cells of the ant Formica polyctena (1045)
and the weta Hemideina maori (1305).
Reabsorption of Water and Ions
Most absorption of nutrients and water occurs across the
midgut in insects. The hindgut then serves to modify what it
receives from two upstream sources: the gut contents following digestion and absorption across the midgut as well as the
fluid secreted by Malpighian tubules. Although the hindgut
performs the bulk of reabsorption of useful ions and water in
most species, a proximal (lower) segment of the Malpighian
tubule modifies the urine of some species. A similar role is
performed by the ileum of the locust, Schistocerca gregaria
(1444). Such reabsorption upstream allows the rectum to process more thoroughly a reduced volume of fluid delivered
to it.
Urine modification by the lower (proximal)
segments of the Malpighian tubules
There appear to be two reasons for reabsorption of water
and/or ions by what is known as the lower or proximal tubule
(i.e., that portion of the tubule’s length closest to the junction
with the gut). For a blood feeder such as Rhodnius prolixus,
the high surface area offered by the lower Malpighian tubule
relative to the volume of fluid in its lumen permits more rapid
reabsorption of KCl during the post-prandial diuresis than
would be possible in the large, sac-like rectum. In Drosophila
and other dipterans, there is extensive reabsorption across the
rectal pads of the hindgut, and the lower Malpighian tubule
probably serves a preparatory or ancillary role in the reabsorption of both K+ and water.
Reabsorption of ions but not water by the Rhodnius tubule
is a consequence of the insect feeding on blood, which is
hypoosmotic to its hemolymph. Homeostasis requires elimination of hypoosmotic urine that is produced in a two stage
process: production of near-isoosmotic primary urine (consisting of approximately 100 mM NaCl, 80 mM KCl) by the
upper secretory segment of the Malpighian tubule, followed
by reabsorption of ions but not water as the secreted fluid
passes through the 30% of the lower tubule’s length closest
to the hindgut (1129). Water is not reabsorbed because the
osmotic permeability of the lower tubule is much lower in
this region relative to the upper tubule or the upper region of
the lower tubule (1331). KCl reabsorption by the lower tubule
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Osmoregulation and Excretion
preserves osmotic and ionic homeostasis; the fluid passing
into the hindgut is both NaCl-rich and hypoosmotic.
The current model of KCl reabsorption suggests that K+ is
first pumped thermodynamically uphill from lumen to cell by
an ATP-dependent transporter that is inhibited by drugs such
as omeprazole, which block the H+ /K+ -ATPase of the vertebrate gastric mucosa. K+ then moves passively from cell to
bathing saline (hemolymph) through an electrodiffusive pathway (i.e., channels) that can be blocked by Ba2+ . The presence
of basolateral K+ channels is indicated by changes in basolateral membrane potential in response to changes in bathing
saline K+ concentration and blockade of these changes and of
K+ reabsorption by Ba2+ . K+ reabsorption is unaffected by
amiloride (which blocks a putative K+ /H+ exchanger in the
upper tubule) and by the V-type-ATPase inhibitor bafilomycin
A1 , applied to the basolateral or apical surfaces of the lower
tubule (685).
Chloride also moves from cell to hemolymph through
channels. Chloride channel blockers such as diphenylamine2-carboxylate and the related compound 5-nitro-2-(3phenylpropylamino) benzoic acid inhibit KCl reabsorption
and also block Cl− -dependent changes in basolateral membrane potential (686). KCl reabsorption is also reduced by
carbonic anhydrase inhibitors such as acetazolamide, consistent with a role for cellular CO2 as a source of both H+
and HCO3 − for exchange of K+ and Cl− , respectively. The
mechanism of Cl− transport from the tubule lumen into the
cell is unclear, although a stilbene-insensitive Cl− /HCO3 −
exchanger has been proposed.
It is worth noting that the lower tubule of adult Drosophila
melanogaster performs multiple functions; it reabsorbs
approximately 30% of the KCl and water secreted upstream
by the main segment of the tubule. In addition, the lower
tubule secretes Ca2+ from the hemolymph into the lumen,
acidifies the lumenal fluid and also secretes organic cations
such as tetraethylammonium at high rates (1344, 1534). In larvae, the lower tubule secretes K+ but reabsorbs Na+ (1287).
The lower Malpighian tubule is thus much more than a simple conduit connecting the fluid secretory main segment to
the ureter and gut. Fluid secreted upstream is also modified
by reabsorptive process in the lower tubule, the ampulla (into
which multiple tubules empty and which is itself connected
to the gut), or both in the house cricket Acheta domesticus
(1745). There are also reabsorptive cells scattered throughout
the epithelia in tubules of the cricket Teleogryllus oceanicus
(1160).
The Insect Hindgut
The hindgut receives the output of the midgut and the
Malpighian tubules; the contents of the hindgut are then modified through reabsorption of useful solutes and water and
elimination of molecules that are present in excess or are
toxic. The hindgut—and the rectum in particular—is thus a
major site of water conservation in insects. An extreme example is evident in the rectum of the mealworm larvae (Tenebrio
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Osmoregulation and Excretion
molitor); water reabsorption is so effective that the fecal pellets that are expelled are almost completely dry. In the hindgut
of stick insects (1501, 1502), water-stressed locusts and cockroaches (1441, 1442, 1924), as much as 80%-95% of the ions
and water secreted by the Malpighian tubules are reabsorbed;
the corresponding figure for blowflies is 65%. When necessary, the extent of water reabsorption by the insect hindgut
can be reduced. When locusts are fed on a succulent diet,
for example, less than 10% of water is reabsorbed from the
hindgut (1099), and blood-feeders such as Rhodnius recover
virtually no water during diuresis.
There appear to be at least four distinct physiological
mechanisms of water conservation in the hindgut of different
species of insect (168, 1439).
Cockroaches, locusts and adult
blowflies—absorption of hypoosmotic
fluid by rectal pads or papillae
In these species there are conspicuous thickenings of the rectal epithelium that are referred to as rectal pads in locusts and
cockroaches and as rectal papillae in adult flies. The processes
of local osmosis and solute recycling during fluid absorption
by the rectal pads of cockroaches were demonstrated in classic work by Wall (1924). Osmotic gradients for absorption
of water from the rectal lumen are produced though active
transport of Na+ , K+ and Cl− . Fluid and ions move from the
rectal lumen into the epithelial cells of the pads and then into
the intercellular spaces between the epithelial cells. The intercellular spaces thus act as a separate compartment from the
hemolymph and the rectal lumen. Salt transport elevates the
osmotic concentration in the intercellular spaces above that
in the lumen, so that water enters by osmosis, distending the
intercellular spaces. Downstream, ions are reabsorbed into the
epithelial cells as the solutes and water move along the intercellular channels. A high flow rate and relatively low osmotic
permeability are presumed to minimize water movement back
into the cells, so that a hypoosmotic fluid is transferred to
the hemolymph. The ions reabsorbed into the cytoplasm can
then be recycled back into the upstream sections of the intercellular spaces so as to permit further osmotic reabsorption
from the lumen. Microsampling of fluid from the rectal tissues shows that fluid in the lateral intercellular spaces near
the lumen is hyperosmotic, and the total ion concentration
in the intercellular channel declines as fluid moves toward
the hemocoel.
The main driver of ion transport in the locust rectum is distinct from the Na+ /K+ -ATPase, which dominates vertebrate
epithelial ion transport and the vacuolar type H+ -ATPase,
which plays a cardinal role in other insect epithelia such as
the midgut and Malpighian tubules. Instead, a primary mechanism of Cl− transport is responsible for absorption of ions
and water. Exhaustive and elegant work by John Phillips and
coworkers has shown that Cl− transport is not coupled to,
or driven secondarily by, movements of Na+ , K+ , HCO3 − ,
Ca2+ or Mg2+ (1439, 1444). An apical V-type H+ -ATPase
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is present and it acidifies the hindgut lumen, but its rate of
transport is only 10%-15% of Cl− -dependent short-circuit
current across the rectum. Regulation of rectal Cl− transport
is accomplished by two stimulatory factors that act through
intracellular cyclic AMP: chloride transport stimulating hormone (CTSH) and ion transport peptide (ITP), as described
below.
Mosquito larvae—secretion of hyperosmotic salt
solutions by rectal salt glands
Hemolymph osmolality in the larvae of saline-tolerant
mosquitoes is ∼300 mosmol/kg, more than three times lower
than that in the surrounding medium. Loss of water by
osmosis across the body surface is compensated by drinking
the medium, but the ingested salt must then be eliminated.
Whereas ions and useful metabolites are reabsorbed across
the anterior segment of the rectum (169, 1784), saltwater
mosquito larvae eliminate excess salt by active secretion of
Na+ , K+ , Mg2+ and Cl− across the posterior rectum from the
hemolymph into the lumen (171).
Tenebrionid beetle larvae—secretion of
hyperosmotic salt solutions within the
cryptonephridial complexes
Survival of larvae of beetles such Tenebrio molitor in
extremely dry conditions depends on effective reabsorption
of water from the rectum before the fecal pellets are eliminated. The structure responsible for water reabsorption is the
cryptonephridial complex, which is comprised of the distal
portions of the Malpighian tubules applied closely to the surface of the rectum by an enveloping perinephric membrane.
The rectum and the tubules thus act in concert. K+ , Cl− and to
a lesser extent Na+ are actively secreted from the hemolymph
into the lumen of each of the six Malpighian tubules. In some
tenebrionid larvae, the concentration of KCl in the tubule
lumen approaches and may even transiently exceed saturation. This “osmotic sink” within the tubule lumen (1121) is
used both for fecal dehydration and for atmospheric water
vapor absorption. The rectal epidermis plays a passive role,
providing osmotic coupling between the rectal lumen and
the tubule lumen. The function of the perinephric membrane
is to provide an osmotic barrier to limit osmotic water flow
from the hemolymph into the complex in response to the elevated osmolality within the Malpighian tubule lumen; instead,
water is extracted from the rectal lumen. Beneath the perinephric membrane and surrounding the tubules is the perinephric space. The activity of K+ in the perinephric space
is close to electrochemical equilibrium with the hemolymph,
whereas the activities of Na+ and H+ are reduced fivefold
and threefold, respectively, below the corresponding Nernst
equilibrium values. These data are consistent with a model
in which cations move from the hemolymph across the perinephric membrane into the perinephric space, which is the
proximal source for cations transported into the tubule cells
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Comprehensive Physiology
and then the lumen (1332). In millipedes, it appears that a
cryptonephric arrangement used for fecal dehydration and
water vapor absorption has evolved independently from that
of the tenebrionids (2038).
Thysanurans (silverfish and firebrats) and flea
larvae—rectal and anal sacs
In thysanurans, there is no evidence for high concentrations of
inorganic salts in the structures implicated in fecal dehydration and water vapor absorption (1333). The anal sac consists
of a highly folded single-layered epithelium. The subcuticular space between the rectal cuticle and the epithelium is
filled with a material that resembles glycerol and also contains mucopolysaccharides. The necessary osmotic gradient
to drive water reabsorption from the lumen appears to involve
dehydration of this material by transport activity of the epithelial cells. Water movement may be produced by electroosmosis (984). In this proposal, a lumen-positive potential of 200
mV is generated by active transport of K+ across the apical membrane of the rectal epithelial cells. It is suggested
that cations are then recycled into the cells through cationselective channels, and that water is coupled electroosmotically to the inward current. Water thus moves first from
the rectal lumen onto the hygroscopic material of the subcuticular space and subsequently into the cytoplasm of the
epithelial cells. The weakness of this proposal is that coupling of ions to water occurs in the subcuticular space at
water activities that are well below the saturation point of
KCl (1333).
In flea larvae, a region of the rectum called the rectal sac
has been implicated in reduction of water activity in the recta
lumen during water vapor absorption (114). The epithelial
cells of the rectal sac are arranged in a ventral and a dorsal
gutter and show a distinct asymmetry along the length of the
organ. Dorsal and ventral gutter epithelial cells show large
numbers of mitochondria associated with deep membrane
infoldings, but whereas the apical membrane is infolded in
the dorsal cells, the basolateral membrane is infolded in the
ventral cells. As for the thysanurans, there is no evidence for
high solute concentrations that might be used to produce colligative lowering of vapor pressure to drive faecal dehydration
and water vapor absorption. The threshold humidity at which
net vapor absorption is feasible is 65% RH, well below the
vapor pressure over saturated solutions of NaCl (75%) and
KCl (85%).
Anal Papillae
The larvae of mosquitoes, midges and fruit flies possess extrarenal organs known as anal papillae that have been implicated
in hemolymph ion regulation and osmoregulation. The papillae are produced during embryonic development, by eversion
of the hindgut tissues. In mosquito larvae, four anal papillae
arise from an extension of the terminal segment and project
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Osmoregulation and Excretion
into the external medium. The walls of the papillae are composed of a one cell thick epithelial syncytium and the lumen
is continuous with the hemolymph. The apical surface of
the papilla is separated from the external medium by a thin
external cuticle. The function of the anal papillae is uptake
of ions, primarily Na+ and Cl− , from the external medium
(442, 1771-1773), thereby contributing to the maintenance of
proper hemolymph ion levels.
In common with other epithelial cells implicated in salt
and water transport, the apical and basal plasma membranes of
the cells of the papillae have many infoldings that are closely
associated with mitochondria (588, 1725). Both morphological and ultrastructural characteristics are consistent with a
role for the papillae in ion uptake from dilute media. The
papillae become longer when larvae are reared in very dilute
water. Conversely, the apical and basal membrane infoldings
decrease and there are fewer mitochondria in the papillae
of larval Aedes aegypti raised in saltwater. Anal papillae of
mosquito larvae also show short term changes in transport
characteristics when the environmental salinity is altered; Na+
and Cl− uptake decrease in higher salinity (441).
The mechanism of transport across the papillae shows
some similarities to the gills of freshwater fish. In larvae of
the midge Chironomus riparius, inhibitors of the Na+ /H+
exchangers (EIPA) and carbonic anhydrase (methazolamide)
provide evidence for Na+ /H+ and Cl− /HCO3 − exchange
mechanisms in the anal papillae. Comparable studies of the
papillae of Aedes aegypti suggest that Na+ uptake at the apical membrane occurs through a Na+ channel that is driven by
a V-type H+ -ATPase, or through a Na+ /H+ exchanger that is
insensitive to amiloride and its derivatives (415). Cl− uptake
occurs through a Cl− /HCO3 − exchanger, with carbonic anhydrase providing H+ to the V-type H+ -ATPase and HCO3 − to
the exchanger. It is suggested that Cl− subsequently crosses
the basal membrane into the lumen of the papilla through
channels, whereas Na+ may be pumped from cell to lumen
via the Na+ /K+ -ATPase. Electron micrographs reveal that the
apical plasma membrane is studded with portasomes, consistent with the presence of the V-ATPase. Immunohistochemical studies indicate the presence of an apical V-ATPase and
a basal Na+ /K+ -ATPase. Transcripts of putative aquaporin
genes are expressed in the anal papillae, which also show
immunoreactivity to a human AQP1-antibody. In conjunction
with observed inhibition of tritiated water uptake in isolated
anal papillae by the aquaporin blocker mercury, it thus appears
that aquaporins faciliate water transport across the cellular
membranes of the papillae (1179).
Neuroendocrine Control of the
Malpighian Tubules and Gut
Most insects spend much of their lives in a state of antidiuresis given that they live in a desiccating environment.
Thus, although the normal condition involves elimination of
relatively dry excreta, excess water is voided after eclosion
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Osmoregulation and Excretion
and also in response to feeding on moist or liquid diets.
Diuretic hormones act in most species by accelerating the production of the primary urine by the Malpighian tubules. Antidiuretic hormones act inhibiting fluid production by the tubules
or, more commonly, by stimulating reabsorption across the
hindgut (1746). The initially paradoxical discovery of diuretic
hormones in desert insects has been explained by noting that
clearing the hemolymph of wastes and toxins requires stimulation of fluid secretion by the Malpighian tubules followed by
downstream reabsorption of water in the hindgut. Factors that
stimulate this process are thus better described as clearance
hormones (1306).
One rationale for studying neuroendocrine control mechanisms for osmoregulatory processes in insects is that such
studies offer potential for development of novel, speciesspecific and environmentally benign control measures for
pest species. Insect kinin neuropeptides, for example, have
been isolated from many species and have been implicated
in control of processes such as hindgut contraction, diuresis and the release of digestive enzymes. Biostable analogs
have been designed so as to protect sites in the pentapeptide kinin sequence from peptidases in the hemolymph and
tissues of insects. In particular, peptide analogs containing
an aldehyde at the C terminus are known to inhibit various
classes of proteolytic enzymes. C-terminal aldehyde insect
kinin analogs lead to fluid retention and mortality in corn earworm larvae and also interfere with diuresis in the housefly
(1268). Peptidomimetic analogs that contain no native peptide bonds but retain significant diuretic activity in an in vitro
cricket Malpighian tubule fluid secretion assay have also been
developed (1267).
The Malpighian Tubules
Factors that alter fluid and ion transport by MTs include
biogenic amines (serotonin and tyramine) and five families of neuropeptides (315, 317, 319, 321). Among the
best-studied peptides are the corticotrophin-releasing factorrelated diuretic hormones (CRF-related DHs) and the kinins,
so named for their effects on hindgut contractions. A third
peptide family is made up of the calcitonin-like diuretic
hormones (CT-like DH). In addition, tubules of locusts and
the hawkmoth Manduca sexta are stimulated by tachykininrelated peptides. The CAPA peptides, products of the capability (capa) gene in Drosophila, are unusual in that they
exert diuretic or antidiuretic effects on tubules of different
species. In dipterans, CAPA peptides elevate production of
intracellular nitric oxide, which, in turn, activates guanylate
cyclase (201, 397, 448, 1125). The increase in cGMP is associated with stimulation of the V-type H+ -ATPase in the apical membrane and a consequent increase in transport of K+
and/or Na+ across the apical membrane (1347). The increase
in ion transport and the flow of osmotically obliged water
results in the increased fluid flow is seen during diuresis. By
contrast, CAPA peptides act as antidiuretics in hemipteran
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tubules, including the blood-feeder Rhodnius prolixus (1385,
1392-1395) and the plant-sucking bugs Acrosternum hilare
and Nezara viridula (322). Antidiuretic factors of the beetle
Tenebrio molitor (Tenmo ADFa and Tenmo ADFb) also lead
to increases in cGMP levels, but do so through mechanisms
that are independent of nitric oxide signaling, and so a soluble guanylate cyclase is unlikely (485, 486, 1993). Tenmo
ADFa and cGMP also inhibit fluid secretion in tubules of
adult Aedes aegypti; the inhibition by ADF does not involve
changes in transepithelial voltage or resistance, suggesting
that electroneutral transport is affected, possibly the basolateral Na+ -2Cl− -K+ cotransporter in the principal cells (1180).
It has been a long-standing question as to why such a multiplicity of factors that alter the Malpighian tubules is needed.
One possibility is that the use of two factors allows for a
synergistic interaction. The net effect of such an interaction
is to steepen the dose-response curve to the point that the
two factors together act essentially as a switch, converting
an unstimulated tubule into a fully diuretic one. Locustakinin
(Locmi-K) and Locusta-diuretic hormone (Locmi-DH), for
example, act synergistically to stimulate secretion by
Malpighian tubules of the desert locust Schistocerca gregaria. When both are present, fluid secretion is stimulated
to a greater extent than the sum of their individual effects.
Kinin effects are mediated through increases in intracellular inositol trisphophate/Ca2+ , whereas those of Locmi DH
are mediated by increases in cAMP. Such synergism would
allow the use of lower concentrations of diuretic factors to be
released, thus minimizing the energetic costs associated with
their synthesis and release. Alternatively, some factors may
be implicated in excretion of specific ions (Na+ versus K+ ) or
in integrating various other epithelia (salivary glands, midgut,
hindgut). Locmi-DH, for example, increases Na+ transport by
locust tubules at the expense of K+ transport in the presence
or absence of locustakinin. Enhanced Na+ transport is also
seen when MTs are stimulated with cAMP. Increased Na+
secretion by Lom-DP acting alone or in synergism with locustakinin may serve to provide sufficient Na+ for use by Na+ coupled cotransporters downstream in the tubule or hindgut
(318). Independent control of Na+ and K+ secretion may
be important, for example, in maintenance of hemolymph
volume and composition after feeding. For the Malpighian
tubules of the tobacco hawk moth Manduca sexta, there are at
least eight different sorts of compounds that significantly alter
the rates of Malpighian tubule fluid secretion, and all of these
factors also accelerate the rate of heartbeat in Manduca and
do so at similar concentrations (1707). To quote the authors of
the latter study, “[T]here may exist in the extracellular fluid a
continuous broadcast of information in the form of a chemical
language, to which many or all parts of the body continuously
respond on a moment-to-moment basis and which, because
of the greater information in it, ensures a more effective and
efficient coordination of function than could be achieved by
a series of single, tissue-specific hormones that force stereotypical responses by their target tissue.”
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Osmoregulation and Excretion
Hindgut and Midgut
Excretion of Toxins
Most of what we know of hormonal control of fluid reabsorption by the insect hindgut is based on studies of locusts, in
part because the large size of the hindgut permits flat sheets
of epithelia to be isolated for use in Ussing chambers. Three
antidiuretic peptides have been isolated from the corpora
cardiaca, a neurosecretory organ analogous to the pituitary
and hypothalamus glands of the vertebrate endocrine system:
neuroparsins (Np), ion transport peptide (ITP) and chloride
transport stimulating hormone (CTSH) (1638). Stimulation
of active absorption of Cl− at the apical membrane by CTSH
or ITP involves cAMP as second messenger.
In locusts, hormonal control of the Malpighian tubules and
the hindgut are clearly separated. Ion-transport peptide (ITP)
released into the hemolymph from the corpora cardiaca stimulates fluid reabsorption by the ileum but has no effect on fluid
secretion by the Malpighian tubules. Conversely, locustakinin
(Lom-K) or Locusta-diuretic hormone (Locusta-DH) stimulate fluid secretion by locust Malpighian tubules but have no
effect on active transport of Cl− or the rate of fluid reabsorption across the ileum or rectum in vitro (320). Responses of
the tubules to diuretic factors are more rapid than the response
of the ileum and rectum to hindgut stimulants such as ITP. As
a consequence, urine flow exceeds fluid uptake in the hindgut
in the early phases of post-prandial diuresis, so excess water
is eliminated.
Many of the neuropeptides present in the neurosecretory
cells of the central nervous system have also been found in
nerve cells within the gut or in midgut endocrine cells (2077).
These findings raise the likelihood that processes such as
absorption of ions and water across the midgut may be modulated by neuropeptides acting as hormones or paracrine factors. Given that secretion of water and ions by the Malpighian
tubules of Drosophila is stimulated by leucokinins and DH31,
it is of interest that receptors for the leucokinins are present
in the midgut of the Drosophila larvae and receptors for
the diuretic hormone DH31 are present in the midgut of
both larvae and adults (1907, 1908). Although stimulation
of midgut ion transport as part of a diuretic strategy has not
been observed in Drosophila, serotonin is well known for its
role in stimulation of both tubule secretion and midgut Na+
and water absorption in Rhodnius (517, 1136), and the CAPA
peptides inhibit both secretion by the tubules and absorption
by the midgut (826, 1391). Although high rates of midgut
ion transport are seen during feeding by larvae of the tobacco
hornworm, there is an abrupt decline in midgut ion transport
when feeding ceases and the larvae enter the wandering stage,
during which it becomes committed to pupation. Possible
hormonal control of this decline in midgut absorption is suggested by the finding that active ion transport across the posterior midgut of the tobacco hornworm is inhibited by Manduca allatotropin (Mas-AT) and by two Manduca FLRFamides
known as F7D (DPSFLRF-NH2 ) and F7G (GNSFLRF-NH2 )
(1027).
Insects have an extraordinary capacity to detoxify and excrete
an enormous range of potentially toxic molecules. This facility reflects, in part, the coevolution of flowering plants and
insects. To minimize herbivory, plants have evolved a wide
range of secondary compounds that are toxic. The presence
of such compounds in the insect diet has lead to selection
for genes that confer some form of resistance to the toxic
molecule. The larva of the tobacco hornworm (Manduca
sexta) can thus survive on a diet containing large quantities
of the cholinergic receptor blocker nicotine, and milkweed
bugs can feed on plants containing high levels of cardiac glycosides, which are potent inhibitors of the Na+ /K+ -ATPase.
This evolutionary arms race between plants and insects has
produced a series of “leapfrog” events, whereby appearance
of a new toxin in a plant’s chemical armoury has led to selection in insects for new enzymes or membrane transporters that
can detoxify or excrete the toxin.
Toxin excretion is accomplished by the Malpighian
tubules and the gut, including the midgut and hindgut. In
the midgut, toxin transporters may play a dual role, minimizing passive uptake of toxins by actively secreting them back
into the gut lumen, or in clearing the hemolymph of toxins.
Exposure of insects to plant-derived or anthropogenic toxins is also associated with increases in the activity of phase
I and phase II detoxification mechanisms. Phase I enzymes
introduce reactive and polar groups into substrates by oxidation, hydrolysis or reduction. Phase I enzymes include the
P450 monoxygenases, which have been implicated to play
a role in metabolism of natural and synthetic pesticides by
insects (529). Metabolites of xenobiotics after phase I reactions may be subsequently conjugated with compounds such
as glutathione, sulphate or glucuronate in phase II reactions.
Glutathione-S-transferases (GSTs) are prominent among the
phase II enzymes; increases in GST levels are correlated with
resistance to all major classes of insecticides (1511). The
phase I and phase II enzymes are found in tissues such as the
fat body and also in the Malpighian tubules. Treatments that
increase P450 and GST gene expression level are also associated both with increases in expression of toxin transporter
genes in the tubules of Drosophila and with toxin secretion
by isolated tubules. Exposure of Drosophila to toxins thus
evokes a coordinated response by the Malpighian tubules,
involving both alterations in detoxification pathways as well
as enhanced transport (266) (Fig. 16).
The hindgut, along with the Malpighian tubules, plays a
primary role in excretion of nitrogenous wastes such as ammonia (as described below in the section on Nitrogen Excretion)
and uric acid. In desert locusts, ammonium urate is excreted:
the urate by the Malpighian tubules and ammonium by both
the tubules and the rectum. This arrangement goes against the
usual view of uricotely in insects but provides multiple advantages. Ammonium urate has a lower solubility than other urate
salts have, so less water is lost when it is excreted, and it also
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437
Osmoregulation and Excretion
Comprehensive Physiology
Hemolymph
X-OH
eI
GS
T
50
P4 ATP
X
X
ADP
Ph
as
as
Ph
X
P-gp
eI
I
GSH
ADP ATP
G-S-X
ATP
MRP
P-gp
ADP
Lumen
Cell
Figure 16 Speculative model showing interaction of detoxification
and excretion pathways in Malpighian tubules and gut. A moderately hydrophobic toxin (X) may be transported unmodified into the
lumen by apical P-glycoprotein (P-gp) and/or multidrug resistanceassociated protein (MRP). Alternatively, the toxin may be modified by
P450 enzymes and either transported into the lumen by P-gp, or further
modified by conjugation with glutathione (GSH) through the actions of
GST and then transported into the lumen by MRP. Toxin X may enter
across the basolateral membrane through active (closed circle) or passive (open circle) mechanisms. Adapted from Bard (71, 1337).
has the additional advantage of providing for elimination of
one additional nitrogen. Exchange of Na+ for NH4 + in the
rectum conserves Na+ , which is in short supply in a herbivorous diet. Moreover, ammonium secretion provides an effective means of increasing acid elimination without increasing
the pH gradient against which the hindgut proton pump must
work. Proton secretion is implicated in recovery of HCO3 − in
the hindgut and possibly in reabsorption of proline (1440).
Transport of organic cations
and organic anions
Both the gut and the Malpighian tubules transport a wide variety of organic ions. Organic ions may be derived from the diet
or metabolism, and many are toxic, thus necessitating elimination from the insect. For both organic cations (OCs) and
organic anions (OAs), there are multiple transporters, often
with overlapping substrate affinities. Most organic cations
(OCs) carry a positive charge at physiological pH and contain
a hydrophobic region and usually a quaternary nitrogen. The
need for multiple transporters is related, in part, to the need to
transport molecules from two broad structural classes. Type
I organic cations are hydrophilic, monovalent and relatively
small (< 400 Da). Examples are endogenous compounds such
as choline and N-methyl nicotinamide (NMN) and drugs such
as tetraethylammonium (TEA) and quinine. Type II organic
cations are larger (> 500 Da), amphiphilic compounds that
contain a positive charge located within or close to a large
aromatic ring structure (2042). Plant toxins such as nicotine
and quinidine are examples of type II OCs.
Much of what is known of type I OC transport in insects
has been the result of studies of secretion of TEA by the
438
Malpighian tubules. Uptake of TEA across the basolateral
membrane has been inferred from microelectrode measurement of basolateral membrane potential and the uptake of
14
C-labelled TEA (1533). Uptake is electrogenic; depolarization of Vbl through increased K+ in the bathing saline reduces
uptake, whereas hyperpolarization of Vbl with lower K+ in
the bathing saline enhances uptake. Addition of TEA to the
bathing saline depolarizes Vbl , consistent with uptake through
a conductive pathway. TEA is not taken up through K+ channels because blockade of K+ channels with Ba2+ does not
alter the effect of TEA addition on Vbl .
TEA-selective microelectrodes based on the cation
exchanger tetra(p-chlorophenyl) borate have been used to
measure transport of TEA by isolated Malpighian tubules
and guts (Fig. 11). This exchanger was originally used for K+
measurement, but its selectivity for TEA exceeds that for K+
by a factor of 10 million. The concentration of TEA in nanoliter droplets of fluid secreted by Malpighian tubules set up in
the Ramsay assay can be measured by placing TEA-selective
and reference microelectrodes in the droplets. TEA secretion
rate (pmol min−1 ) is then calculated as the product of fluid
secretion rate (nl min−1 ) and secreted fluid TEA concentration
(mmol l−1 ). It is worth noting that the contribution of transporters to elimination of toxins by the Malpighian tubules cannot be unequivocally determined using only measurements of
lumen to bath ratios of toxin concentration. Changes in the
rates of fluid secretion rate influence the concentration of
the solute in the lumen, particularly if the permeability of
the tubule wall to the solute is high. A stimulant of fluid
secretion will thus appear to inhibit transport of a substrate if
only lumen-to-bath concentration ratios are measured, since
the concentration of the solute in the lumen will be reduced.
In fact, the transepithelial flux of the solute may actually
increase, since the reduction in lumenal concentration will
reduce the extent of passive diffusional backflux of the solute
from lumen to bath. For this reason it is essential to measure
both the concentration of the transported solute in the lumen
and the fluid secretion rate, so that the transepithelial flux may
be calculated.
This reservation applies for measurement of toxin secretion using TEA-selective or salicylate-selective microelectrodes (described below), and also when OC or OA transport
is measured by the concentrations of fluorescent transport
substrates using fluorescence or confocal microscopy. Some
transport substrates are associated with increases or decreases
in fluid secretion rate. Rhodamine 123, for example, inhibits
fluid secretion rate at concentrations well below the concentration associated with half the maximal transport rate (Kt ).
Thus, although the luminal concentration is high, the rate of
transport is quite low. Measurement of luminal fluorochrome
concentrations is complicated by quenching, reflection or
absorption of the exciting and/or emitted light by the tissues and by opaque uric acid crystals present in the lumen.
It is not possible to clearly visualize fluorochromes and the
lumen of Malpighian tubules of Drosophila tubules because
of opaque concretions in the cells and/or lumen (1023).
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Comprehensive Physiology
Further complications arise when the concretions move in
the lumen during fluid secretion, thereby altering the amount
of laser light passing through the lumen, or when concretions
may be transferred from the cell to the lumen, particularly
when fluid secretion is stimulated. Collection of secreted fluid
droplets after concretions have settled allows a clean sample
of secreted fluid to be collected, and fluorochrome concentration can be measured by taking a sample of the secreted fluid
up in a rectangular glass capillary that is optically flat, allowing the fluorochrome concentration to be readily measured by
confocal laser scanning microscopy.
TEA-selective microelectrodes can also be used in conjunction with the scanning ion-selective electrode technique to
measure TEA fluxes across specific regions of the Malpighian
tubules, the ureter that connects the tubules to the gut in some
species, and across the gut itself. In Drosophila, TEA is transported at high rates by the proximal segment that also reabsorbs KCl and water and at lower rates by the fluid secreting
main segment, but not by the distal, non-fluid-secreting distal segment that sequesters Ca2+ as luminal concretions. In
the Ramsay assay, the concentration of TEA in the lumen of
the main segment is ∼12-fold higher than that in the bathing
saline. Given that the lumen is at an electrical potential of
30 mV to 80 mV positive to the bath, TEA secretion is a thermodynamically active process. TEA uptake is competitively
inhibited by other type I OCs, including other quaternary
ammonium compounds, cimetidine and quinine. TEA uptake
is also inhibited by the type II OCs verapamil and nicotine,
suggesting that the basolateral OC transporter has a very broad
substrate specificity.
Alteration of excretory mechanisms in response to
dietary exposure to toxins
An increased capacity of the Malpighian tubules to secrete
toxins is seen after dietary exposure of insects to the same
or related toxins. The adaptive value of such an increase is
presumably that the insect does not incur the high metabolic
cost of maintaining transporters that are not required in the
absence of toxin exposure.
When larval Drosophila are exposed to 100 mM TEA
in the diet, the concentration in the hemolymph increases to
∼ 3 mM. Malpighian tubules isolated from larvae reared on
diet containing 100 mM TEA secrete TEA at rates approximately 77% higher than those seen in tubules isolated from
larvae reared on a TEA-free diet (132). Clearance of TEA
from the hemolymph is slowed if the larvae are exposed simultaneously to TEA and cimetidine, a competitive inhibitor of
TEA transport by the tubules and posterior midgut.
Expression of P-glycoproteins (P-gp’s) in insects is also
influenced by dietary exposure to transporter substrates. Both
the gut and the Malpighian tubules are important sites of
P-gp expression in insects. In D. melanogaster, P-gps are
the products of expression of three genes, MDR49, MDR50
and MDR65. Increased MDR expression is evident in all
sections of the gut (anterior and posterior midgut, hindgut
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Osmoregulation and Excretion
and Malpighian tubules) when larvae of D. melanogaster are
fed diets containing a non-toxic level of the P-gp substrate
colchicine (10 micromolar), relative to control larvae raised
on colchicine-free diet (1807).
By contrast with organic cations, dietary exposure to
organic anions results in effects on both rates of OA transport and also the rate of fluid secretion (1589). Because fluid
secretion is a passive osmotic consequence of secretion of
inorganic ions (Na+ , K+ , Cl− ) into the tubule lumen, these
findings indicate that exposure to dietary organic anions leads
to increased expression or activation of transporters such as
the V-type H+ -ATPase, which energizes transepithelial ion
transport. Salicylate influx across the posterior midgut, ileum
and rectum is not altered in larvae chronically exposed to
dietary salicylate. Thus, absorption by the gut is not downregulated in response to excess dietary salicylate. Instead, excretion is enhanced by upregulation of Malpighian tubule salicylate transport. Measurement of Michaelis-Menten parameters
in tubules of larvae reared on salicylate-rich diets suggest that
transport rate is increased by an increase in the number of
transporters (as seen in the increase in the Michaelis-Menten
parameter Jmax from 5.8 pmol min−1 tubule−1 in tubules from
larvae on the control diet to 28.1 pmol min−1 tubule−1 in
tubules from larvae fed salicylate-rich diets). It appears that
these transporters are different than those present in the controls, because transport shows a lower affinity for salicylate
(Kt = 0.42 mmol l−1 ) relative to tubules from larvae reared
on the control diet (Kt = 0.09 mmol l−1 ) (1589).
Malpighian tubules isolated from larvae reared in
salicylate-rich diets larvae are apparently modulated through
the insertion of more ion transporters into the cell membranes,
rather than through an increase in the basal level of intracellular second messengers (cAMP or Ca2+ ) for diuretic factors.
An increase in the number of V-type proton pumps in the
apical membrane of the principal cells will therefore lead to
an increase in secretion of both cations (Na+ and K+ ) and
Cl− , with a consequent increase in the rate of fluid secretion.
Exposure to dietary toxins may thus result in a remodeling of
the epithelium, with more and/or different transporters produced by transcriptional, translational or post-translational
events. Such phenotypic plasticity is also seen in response
to variations in dietary salt intake for Drosophila larvae
(1287, 1288) or to alterations in ambient salinity for mosquito
larvae (443).
Type I organic anions are particularly effective in producing changes in fluid secretion rate and OA transport rate.
Exposure to D. melanogaster larvae to salicylate-rich or
fluorescein-rich diets for only 24 hours is sufficient to cause
a nearly twofold increase in fluid secretion rate and in salicylate transport, but also in the tubule secretion of methotrexate,
a type II OA (265). MTX transport is Na+ independent and
has distinct transport kinetics (lower Kt , lower Jmax ) relative
to salicylate transport, so different transporters are involved.
By contrast, fluid secretion rate and MTX secretion increases
after 10 days of exposure to dietary MTX, and there is no associated increase in salicylate secretion. These findings suggest
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Osmoregulation and Excretion
a preeminent role for type I OAs in signaling the increased
basal rate of fluid secretion and OA transport rate (265). Much
of the increase in the rate of salicylate or MTX secretion
is a response to an increase in fluid secretion rate. Tubules
of Drosophila have a relatively high permeability to these
molecules, and so there is a tendency for passive backflux in
response to the high concentrations in the lumen and the lower
concentrations in the hemolymph. A twofold increase in fluid
secretion rate will thus halve the concentration of the OA in
the tubule lumen, and there is a consequent reduction in the
gradient driving passive backflux. The net effect is an increase
in secretion of the OA by the tubule. A similar response is
seen when fluid secretion rate is increased in response to
addition of diuretic factors or their second messengers; the
reduction in diffusive backflux results in increased net secretion of organic anions (1343). It is important to emphasize
that this phenomenon is dependent on a relatively high permeability of the tubule wall to small molecules. Tubules of
the blood-feeding hemipteran Rhodnius have a much lower
permeability to small molecules, so that there is little diffusive backflux, and changes in fluid secretion rate do not alter
the rates of passive or active secretion of small molecules by
the tubule (1131).
It is worth noting that a very different strategy of dealing
with potentially toxic material is seen in the midgut of lepidopteran and mosquito larvae. The plant detritus on which
the larvae feed contains tannins, which are potentially toxic
because they complex freely with enzymes, thereby disrupting insect digestion by immobilizing gut enzymes via nonspecific blockage of active sites (109). High pH in the lumen
of the anterior midgut favors dissociation of tannin-protein
complexes and is also optimal for digestive enzyme function
in the larvae. An intriguing aspect of midgut physiology of
larval mosquitoes is the presence of the regional variations
in luminal pH. From near-neutrality in the foregut, luminal
pH increases in the anterior midgut to >10, one of the highest recorded values in animals, then declines to 7.5 in the
posterior midgut.
Much of our knowledge of the physiological mechanisms
of alkaline pH generation in the midgut lumen is derived
from the extensive studies by Moffett, Onken and coworkers
(1376). Lumenal alkalinization is energized by a V-ATPase
in the basal membrane (1407) transferring protons from cell
to hemolymph. The neurohormone serotonin stimulates the
V-ATPase activity, leading to surprisingly high intracellular
pH values (8.58) (1377). However, the means by which acidic
equivalents are transferred across the apical membrane (from
lumen to cell) are much less clear. Inhibitor studies suggest
that carbonic anhydrase is not involved in supplying acidbase equivalents to the respective transporters. Surprisingly,
the Na+ /K+ ATPase is present in the apical (lumen-facing)
membrane of the gut but appears not to be involved in driving luminal alkalinisation (1378), which is maintained in the
presence of ouabain. Pharmacological studies also rule out
the involvement of apical Cl− /HCO3 − exchangers, apical H+
channels and apical cation/proton exchangers (1376).
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Comprehensive Physiology
Transport and excretion of divalent ions
and bicarbonate
Calcium Insects regulate the level of Ca2+ in their extracellular fluids by a mechanism fundamentally different from that
operating in vertebrates. Calcium homeostasis in mammals
results from precise control of transcellular calcium absorption by the duodenum. By contrast, control of excretion and
not absorption is the major means of hemolymph calcium
regulation in flies (Calliphora vicina) (1810). In response
to Ca-rich diets, the calcium content of Ca. vicina rapidly
increases to more than double the original content but then
remains stable. However, midguts isolated from flies fed either
Ca-rich solutions or water absorb calcium at the same rate.
It appears that rapid calcium absorption from the blowfly
midgut is necessary to remove calcium from the lumen so
that other processes such as phosphate absorption can operate. Regulation of hemolymph Ca2+ is accomplished not by
adjustments of midgut absorption but through increases in the
rate of Ca2+ removal by the Malpighian tubules. The tubules
of dipterans remove calcium from the hemolymph through the
processes of secretion and sequestration. Approximately 85%
of calcium that enters the tubule is sequestered, and 15% is
secreted in soluble form into the tubule lumen. Although adult
fruit flies may ingest 30 times more calcium than they retain,
hemolymph calcium concentration and whole animal calcium
content are precisely regulated. A 620% increase in dietary
calcium level, for example, is associated with an increase
in whole fly calcium content of only 10%, and hemolymph
calcium concentration (∼0.5 mM) is similar in flies raised on
diets differing more than sixfold in calcium content (454). The
main segment of the Malpighian tubule of D. melanogaster
contributes to this regulation by secreting an amount of Ca2+
equivalent to the entire body Ca2+ content in approximately
9 hours. Much more rapid elimination of calcium is achieved
through sequestration of Ca2+ as concretions of bicarbonate
and phosphate in the distal segment of the anterior pair of
tubules. The two distal segments can sequester an amount of
calcium equivalent to the body Ca2+ content in approximately
2 hours (454).
Secretion and sequestration of calcium by the tubules are
independent processes. Ca2+ sequestered within the tubule
is released into the lumen even when basolateral uptake is
blocked. Whereas cAMP increases both basolateral uptake
and transepithelial secretion of Ca2+ , the calcium channel
blockers diltiazem and verapamil inhibit only basolateral
uptake (453). A role for Ca2+ uptake through channels is
suggested by increases in uptake of Ca2+ across the basolateral membrane when basolateral membrane potential is
hyperpolarized by bathing tubules in saline with reduced K+
concentration. Transport from cell to lumen is thermodynamically uphill against opposing chemical and electrical gradients
and must involve some form of Ca2+ pump, either an ATPase
or an electrogenic exchanger (e.g., 3Na+ :Ca2+ ). Transepithelial Ca2+ secretion is increased by treatments that depolarize
the transepithelial potential by increasing Cl− conductance.
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Comprehensive Physiology
Exposure to bicarbonate-free saline acidifies the secreted fluids and also enhances transepithelial Ca2+ secretion, possibly through increased dissolution of intracellular granules
through changes in intracellular pH.
By contrast with dipteran tubules, the Malpighian tubules
of Rhodnius sequester almost all of the Ca2+ ingested (1139).
Avian blood contains 1.5 mM Ca2+ , and the insect ingests a
blood meal equivalent to ∼10 times its unfed weight, so Ca2+
is present in considerable excess. The rationale for sequestration rather than secretion may be to avoid exposure of hindgut
transporters to high levels of Ca2+ , which might interfere with
rectal ion and water transport. Alternatively, calcium deposits
in the tubules may subsequently be mobilized for production
of eggs or spermatophores after ecdysis of adults. Rates of
calcium uptake by the tubules increase several days after the
blood meal, coincident with release of Ca2+ from the digested
red blood cells. The post-prandial increase in tubule Ca2+
uptake may be an example of an inducible transport system,
stimulated by some correlate of feeding. Similar increases in
tubule secretion of organic anions such as para-aminohippuric
acid and uric acid are also seen after the blood meal. It is also
worth emphasizing that the calcium concretions in the tubules
are dynamic structures rather than static precipitates. 45 Ca that
has been incorporated in the previous 48 hours into the tubules
is released at the rate of about 25% per hour when the tubules
are transferred to 45 Ca-free saline. Sequestration of calcium is
thus a continuous and metabolically dependent process. Calcium becomes less accessible to the hemolymph over time,
presumably since the “oldest” calcium is near the center of
the concretions. Tubules of larval Rhodnius and crickets are
relatively free of intracellular Ca2+ concretions immediately
after ecdysis, suggesting that the concretions are eliminated
during the moult cycle. Cricket tubules also transfer intracellular calcium concretions into the lumen in response to
stimulation with cAMP, a second messenger for CRF-related
diuretic hormones (721).
Bicarbonate An extreme example of bicarbonate excretion is seen in the rectal salt gland of the larvae of the mosquito
Aedes dorsalis. The larvae are found in alkaline (pH 10.5)
lakes containing high levels of bicarbonate (250 mM) and carbonate (100 mM). Although filter feeding and replenishment
of water lost by osmosis across the integument entail ingestion of the medium at rates of 130% body weight per day,
the larvae maintain hemolymph bicarbonate concentration at
8-18.5 mM and hemolymph pH at 7.55-7.70 (1783-1785).
It is suggested that the salt gland normally secretes a NaClrich fluid into the lumen; the posterior and anterior segments
secrete approximately 75% and 25% of the total, respectively.
The anterior segment of the rectal salt gland is involved in Cl−
uptake (via Cl− /HCO3 − exchange). Bicarbonate and carbonate are excreted into the lumen of the posterior rectum against
both chemical and electrical gradients. However, both salt
gland segments are capable of secreting strongly hyperosmotic fluids containing high concentrations of Na+ , Cl− , and
HCO3 − -CO3 2− (169-172, 1785). The fluid is then modified
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Osmoregulation and Excretion
by ion exchange and reabsorption processes to suit the osmoand ion regulatory requirements of the larva.
Toxic metals
Insects are relatively insensitive to cadmium, copper, lead,
nickel and zinc in acute laboratory toxicity tests, with LC50
values typically about four orders of magnitude higher than
concentrations found in nature (199). Populations of Chironomus riparius that have adapted to high levels of cadmium in
the water both accumulate more cadmium and show higher
rates of cadmium elimination when transferred to cadmiumfree water (1472). Cadmium is first sequestered by metalbinding proteins, then stored in the form of membrane-bound
concretions in the posterior midgut of Chironomus and subsequently expelled into the lumen by exocytosis or degeneration
of whole cells (352, 1645). The gut does not act as a complete barrier, allowing access of Cd to the hemolymph (1033).
The anterior midgut is the main site of Cd entry into the
hemolymph, whereas the posterior midgut absorbs Cd from
the gut lumen as well as from the hemolymph. Cadmium is
also both secreted by and sequestered within the Malpighian
tubules. Although sequestration is the major mechanism at
high levels, secretion is the dominant form of detoxification
by the Malpighian tubules at levels closer to those that are
environmentally relevant (10 μmol l−1 or less). Experiments
with isolated tissues suggest that movement of Cd into the
hemolymph across the anterior midgut is balanced by movement of Cd toward the lumen by the remaining gut segments
and by secretion and sequestration by the Malpighian tubules.
As a consequence, levels of Cd do not tend to increase further.
Although high doses of Zn and Cu are toxic, both metals
are components of many enzymes, and their levels in tissues
of the fruit fly Drosophila represent regulated storage rather
than deposit excretion. Zinc is accumulated primarily in the
main segments of both the anterior and posterior Malpighian
tubules at concentrations up to 2.8% of tissue dry weight
(1637).
Limiting Respiratory and Cuticular Water Loss
The success of insects in the terrestrial environment is something of a paradox given that their small size and consequently large ratio of surface area to volume should render
them vulnerable to desiccation. An appreciation of the desiccating stresses imposed on small terrestrial animals such
as insects can be gained by comparing exposure to subsaturated air with exposure to the osmotic pressures experienced
in hypersaline aquatic environments. The osmotic pressure
(OP) in mosmol/kg equivalent to a specific % relative humidity (%RH) is given by the equation:
OP = RT/V(ln(%RH/100)) = 55(ln(%RH/100)) × 103
Vertebrate body fluids (∼300 mosmol/kg) are thus in equilibrium with 99.5% RH, and seawater is in equilibrium with
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Osmoregulation and Excretion
98.2%RH. The Dead Sea (∼33.7% salinity; ∼ 10 osmol/kg)
provides an osmotic pressure approximating 90% RH.
Given these extraordinarily desiccating conditions in the
terrestrial environment, there has been strong selection pressure for limiting water loss during the evolution of insect life.
Water can be lost in multiple ways: through the respiratory
system by evaporation through open spiracles; through water
associated with elimination of urine or feces or present in
eggs or cocoons; or by transpiration across the cuticle of the
exoskeleton.
Cuticular water loss
Insects provide examples of some of the most impermeable
body surfaces known, and desert species such as tenebrionid
beetles minimize transpiration in spite of exposure to high
ambient temperature and low relative humidity. Although respiratory water loss is significant in active insects, more than
80% of water is typically lost across the cuticle (607). The
primary barrier to evaporative water loss through the cuticle
is a thin (< 1 um) layer of epicuticular lipids. The lipids
consist primarily of hydrocarbons in most species, but wax
esters, ketones, alcohols and sterols have also been identified. The importance of lipids in providing a barrier to water
loss is evident in the 10-100-fold increase in water loss rate
after surface lipids are removed by brief exposure to organic
solvents (683). Although solvents may also alter respiratory
water loss, cuticular transpiration can be isolated by measuring water loss when CO2 release is negligible (i.e., when the
spiracles are closed). The phase transition model proposes that
cuticular lipids exist in a solid state at moderate temperatures
and can thereby form an effective barrier to cuticular transpiration (606). However, the lipids melt and become more
permeable as temperature increases; as a consequence, water
loss also increases rapidly. The n-alkanes in insect cuticle
typically have chain lengths of 20-40 carbons. Insertion of
cis double bonds or addition of one or more methyl groups
will alter both the melting point and water permeability. In
addition, some cuticular lipids are more polar and contain
alcohols, aldehydes, ketones and wax esters. In general, one
might expect increases in hydrocarbon chain length within
or between species to be associated with decreases in permeability, on the assumption that water molecules must diffuse
across the hydrophobic interior of a lipid layer during transporation. A priori, one might also expect that increasing the
degree of unsaturation would decrease the effectiveness of
molecular packing and thus lead to an increase in water permeability. However, experimental evidence shows that chain
length is the least important factor affecting the melting point
of cuticular lipids; methyl branching or insertion of ester linkages or cis double bonds tends to have far more dramatic
consequences. The effects of methyl-branching depend on
the position of the branch point; shifting the branch from a
terminal to an internal position decreases Tm (604, 606).
Most insects have complex mixtures of cuticular lipids;
unfortunately, we know relatively little about how different
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Comprehensive Physiology
compounds interact to determine cuticular permeability. However, Fourier transform infrared spectroscopy (FTIR) can be
used to analyze the temperature dependency of lipid phase
behavior, and such studies allow inferences to be drawn about
the effects on water permeability. Studies of two component
mixtures of hydrocarbons by FTIR do not reveal biphasic
melting transitions expected on the basis of independent phase
behavior of the component lipids. Instead, melting occurs over
a broader temperature range than pure hydrocarbons (603)
and is associated with increased cuticular water permeability
(605, 1581).
Because transpiration increases dramatically above a
species-specific “transition temperature” in some insects, it
has been suggested that there might be a corresponding phase
change in the cuticular lipids. An abrupt change in slope of
a plot of water loss rate as a function of temperature was
interpreted as an indication of a temperature-induced alteration of cuticle structure. The point where the slope changes
is referred to as the critical transition temperature (CTT) in
Arrhenius plots of the logarithm of water loss rate (WLR)
against the reciprocal of temperature (T):
ln WLR = −Ea /RT + ln A,
The slope (−Ea /R) is the activation energy (Ea ) divided by
the gas constant (R) and A is the intercept. In the context of
water loss from an insect, Ea is an indicator of the amount of
energy that a water molecule needs to pass through the cuticle. Plotting the logarithm of WLR is essential, since apparent
CTTs in untransformed data may disappear when the data are
displayed in an Arrhenius plot (2054). More importantly, the
removal of lipids from the cuticle using solvents or physical
abrasion may shift the Arrhenius plots upward on the ordinate
but does not change the CTT or Ea . The upward shift on the
ordinate is consistent with enhanced water diffusion across
the cuticle when the lipids are removed. These findings show
that the Ea for water loss on the Arrhenius plots is not a true
activation energy, and that the CTT appears unrelated to lipid
structure. It is still unclear as to whether the transition temperature for permeability is related to some form of lipid phase
transition, or whether partial melting is sufficient to allow
increased permeability (603). Early literature also suggested
that a specific physical arrangement of lipid molecules in the
cuticle, such as an orientated monolayer, might contribute
to the reduced water permeability. However, use of a magnetic resonance technique suggests that lipids are unlikely to
interact with the cuticle so as to form a monolayer, since the
epicuticular lipids do not show a preferred orientation (1842).
In summary, surface lipids are important in determining
cuticular transpiration, but other factors have a role to play.
The cuticle is a complex structure, and aspects such as the
degree of melanization may also alter permeability. Melanin
granules are polymers of dopa and other tyrosine derivatives
that darken the cuticle. However, melanin may also reduce
the permeability of the cuticle because it is hydrophobic.
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Comprehensive Physiology
Respiratory water loss
The respiratory system of insects consists of a ramifying system of tubes (trachea) terminating in tracheoles that may
extend into cells and lie against the mitochondria. The tracheal
system communicates with the atmosphere through valve-like
structures called spiracles. In many insects and other arthropods, the pattern of exchange of respiratory gases between the
tracheal system and the atmosphere is discontinuous. These
discontinuous gas cycles (DGCs) are based on a pattern of
opening and closing of the spiracles.
DGCs generally comprise three phases: closed (C), flutter
(F) and open (O). When the spiracles are tightly closed, oxygen consumption by the tissues reduces the partial pressure of
oxygen (pO2 ) within the tracheae. The buffering capacity of
the hemolymph and the 24-fold to 30-fold higher solubility
of CO2 relative to O2 means that there is not a corresponding
increase in gas pressure exerted by CO2 . As a result, pressure
within the tracheal system drops below that in the atmosphere.
When pO2 declines to a level of ∼2-4 kPa, the flutter phase
begins. The spiracles transiently open partially, then close in
quick succession; water loss is thought to be limited during
F phase because the water vapor that is tending to diffusing
outward is entrained in the bulk inward movement of air produced in response to the negative pressures within the tracheal
system. Carbon dioxide continues to accumulate within the
tracheal system throughout the F phase. When endotracheal
pCO2 reaches a level of ∼3-6 kPa, the spiracles open widely,
essentially allowing the accumulated CO2 to be flushed out as
the tracheal gases to equilibrate with the atmosphere. Ventilatory movements may aid diffusive gas exchange during the
O phase.
Several hypotheses have been advanced to explain the
adaptive value of DGCs (1182). The three best known are:
(1) the hygric hypothesis, which suggests that DGCs reduce
respiratory water loss; (2) the chthonic hypothesis, which suggests that DGCs facilitate gas exchange during environmental
hypoxia, hypercapnia or both; and (3) the oxidative-damage
hypothesis, which suggests that DGCs minimize oxidative
tissue damage.
Each of the models makes specific predictions regarding
the lengths of DGCs. The respiratory surfaces are saturated
with water vapor, so rates of water loss will be proportional to
the saturation deficit, which is influenced by the temperature
and water vapor pressure of ambient air. Because saturation
deficit is highest at high temperatures and low ambient water
vapor pressures, the hygric hypothesis predicts a positive relationship between DGC duration and ambient temperature, and
a negative correlation between DGC duration and precipitation. The chthonic hypothesis predicts longer DGC duration
in hypoxic habitats. For the oxidative-damage hypothesis, low
ambient pO2 will result in low endotracheal pO2 values, thus
reducing the need for a prolonged C phase to reduce endotracheal pO2 , and a consequent increase in DGC duration. The
oxidative damage hypothesis thus predicts a positive association between pO2 and DGC duration.
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Osmoregulation and Excretion
A meta-analysis based on published data for 40 wildcaught species revealed a significant positive relationship
between DGC duration and habitat temperature and an important interaction between habitat temperature and precipitation.
Based on these results, it appears that the hygric hypothesisis is supported and that DGCs of insects reduce respiratory
water loss but allow for adequate gas exchange (1978). It has
also been proposed that DGCs reflect circadian, developmental or artificially induced reductions in brain activity. DGCs
may result when the thoracic and abdominal ganglia regulate
ventilation in the absence of control from higher neural centers, possibly corresponding to a sleeplike state in the insect
(1182).
Water Vapor Absorption
The small size of insects and the resulting large ratio of surface
area to volume mean that they are in danger of desiccation
in the terrestrial environment. Water is lost by transpiration
across the body surface, through respiration and through elimination of wastes by the gut. Given the potential for water loss
on land, it is perhaps counterintuitive that insects have become
the dominant terrestrial group of animals in terms of numbers
of species and individuals. In addition to sophisticated mechanisms for reducing cuticular and respiratory water loss, some
insects are able to gain water by condensing atmospheric
water vapor. Flying insects can readily and quickly access
water sources separated by distances that are large relative
to the insect’s dimensions. For many non-flying species or
larvae, however, the distances between liquid water sources
may be prohibitive; water sources only a few tens of meters
distant may be unobtainable for a slow-moving species that is
only a few millimeters in length. But although sources of liquid water may thus be unobtainable, the high rates of gaseous
diffusion mean that the insect is continually exposed to water
in the form of vapor. The importance of water vapor absorption as a terrestrial adaptation is evident in the independent
evolution of this phenomenon at least nine times. Different
sites and fundamentally different mechanisms are involved,
as seen in the examples listed in Table 5.
The repeated evolution of WVA mechanisms may be
related, in part, to the relatively low metabolic cost of the
process, even in the face of thermodynamic gradients that
are very large. The gradients can be appreciated by expressing the minimum % relative humidity (%RH) at which water
vapor is absorbed with an equivalent osmotic concentration
(Cosmol , osmol/kg) using the equation given above in the first
paragraph of the section “Limiting Respiratory and Cuticular Water Loss.” For an insect such as the desert cockroach,
absorbing water vapor at 82.5%RH, the equivalent osmotic
concentration is 11.8 osmol/kg, approximately 10 times that
produced by electrolyte concentration in the loop of Henle in
the human kidney. For species such as the firebrat Thermobia, absorbing water vapor at 43%RH, the osmotic gradient
is > 70 osmol/kg.
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Table 5
Comprehensive Physiology
Sites and Mechanisms of Water Vapor Absorption in Terrestrial Arthropods
Species
Site
Proposed Mechanism
References
Desert cockroach (Arenivaga
investigata)
Mouthparts (Hypopharynx)
Hydrophilic cuticle
(1330, 1335, 1336, 1339)
Ticks (Ixodes ricinus)
Mouthparts (Hypostome)
Hydrophilic cuticle
(572)
Booklice (Psocoptera)
(Ctenolepisma sp.)
Mouthparts
CLVP: Labial glands
(1585)
House dust mites
Coxal glands
CLVP: Hyperosmotic
secretion (NaCl, KCl)
(50)
Millipede (Polyxenus lagurus)
Cryptonephridial complex
CLVP: Hyperosmotic
secretion
(2038)
Mealworm (Tenebrio molitor)
Cryptonephridial complex
CLVP: Hyperosmotic KCl
(1122, 1332, 1505)
Flea (Xenopsylla cheopis)
Rectum
Unknown
(114)
Thysanurans (Thermobia
domestica)
Evaginated rectal sacs
Electro-osmosis
(984, 1324)
Isopods (Porcellio scaber)
Abdominal pleopods
CLVP: Hyperosmotic NaCl
(2037)
Collembolans
Body surface
CLVP: Sugars and polyols
(793)
Abbreviation: Colligative lowering of vapor pressure (CLVP)
In spite of the large gradients involved, the metabolic
cost of WVA is comparatively low; absorption of 6% body
weight per day by the desert cockroach requires an energy
expenditure equivalent to less than 1.5% of the insect’s basal
metabolic rate (474). Similarly, absorption by Thermobia for
1 hour requires metabolism of as little as 18 μg of glucose, or
less than 0.07% of the body weight. For comparison, flying
insects may consume fuel at a rate of 0.6% to 20% of the body
weight per hour (1128).
Mechanisms of WVA proposed for most species rely
on colligative lowering of vapor pressure at a specific site
through production of highly concentrated organic or inorganic solutes. In these schemes, energy is required for concentrative solute transport, atmospheric water vapor condenses
and the solutes are recycled after the condensed water is transferred into the hemolymph.
The rectal complex of tenebrionid beetle larvae provides
the best characterized example of solute-coupled WVA. The
species that have been studied include the common mealworm
larvae of Tenebrio molitor as well as the larvae of several
Namibian desert species in genus Onymacris. The adults of
the Namibian beetles derive water from coastal fogs. Adult
Onymacris unguicularis, for example, collect dew on their
dorsal body surface and conduct it to the mouth; other species
collect fog water droplets that have condensed on vegetation,
or collect the water that condenses on the raised ridges of
trenches they have dug in the sand (690).
In contrast to the collection of fog water from saturated
or near-saturated humidities by adult Onymacris, several
species of tenebrionid beetle larvae can absorb water vapor
from humidities as low as 84% RH in the case of Onymacris
marginipennis (1122). The driving force for WVA is the
444
production of a highly concentrated solution of KCl within
the lumen of the Malpighian tubules, as determined in the
original micropuncture measurements of Ramsay (1505) and
subsequent measurements of lumenal [Na+ ], [K+ ], [Cl− ]
and pH using ion-selective microelectrodes (1122, 1332).
WVA is accomplished by the rectal complex, which consists
of the distal ends of the Malpighian tubules applied to the
outer surface of the rectum and enveloped in the perinephric
membrane. The multiple layers of flattened cells that form
the perinephric membrane provide a water-impermeable
barrier between the high solute concentration the Malpighian
tubule lumina and the hemolymph. As a consequence, water
or water vapor within the rectal lumen is at a higher activity
than within the Malpighian tubule lumina. Water flows across
the intervening rectal epidermal cells, which act as a passive
osmotic coupling, and into the lumen of the tubules. The
osmotic gradient established within the rectal complex is
involved both in recovery of water from the fecal pellets
within the lumen and atmospheric water vapor absorption.
Electrochemical gradients calculated from measurements
with ion-selective microelectrodes indicate that the proximal
source for ions transported into the tubule lumen is the
perinephric space. Ions may pass from the hemolymph to the
perinephric space through the thinner and more permeable
anterior perinephric membrane (1333).
Most WVA mechanisms depend on active epithelial ion
transport to produce concentrated solutions with the required
reduction of water vapor pressure. In tenebrionid beetle larvae, for example, condensation of atmospheric water vapor
in the rectum is a passive and secondary consequence of primary active salt secretion into the lumen of the Malpighian
tubules of the rectal complex. Energy is thus required to
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Comprehensive Physiology
drive ions thermodynamically uphill from lower concentrations in the hemolymph to higher concentrations in the tubule
lumen.
In the desert cockroach, however, WVA appears to be a
passive consequence of the hydrophilic properties of specialized mouthparts, and energy is required to effect the release of
water after condensation. Water vapor is absorbed at humidities above 82.5% RH. An adult female can absorb 20 mg day−1
at 96% RH, equivalent to about 4% of body mass per day. During vapor uptake, atmospheric water vapor condenses onto
two bladder-like diverticula of the hypopharynx that protrude
from the mouth. The uptake is measured as weight gain when
an animal is placed in a continuously recording electronic
microbalance under conditions of controlled temperature and
humidity. Surface temperatures of the bladders, measured by
miniature thermocouples, are warmer than the surrounding
mouthparts. This temperature difference is produced by heat
release during the vapor-liquid phase change during WVA
and is humidity dependent (1339). Greater condensation in
higher humidity results in increased rates of weight gain and
higher bladder temperatures.
A pair of non-epithelial structures situated beneath the
frons in the head produces a fluid applied to the bladders
during WVA. This fluid is an approximately isoosmotic ultrafiltrate of the hemolymph and does not provide an “osmotic
sink” to drive atmospheric WVA. The frontal bodies that produce the fluid are thickened derivatives of the integument.
Cyclical contractions of an enveloping muscle mass distort
the frontal bodies and produce internal pressure changes sufficient to extrude fluid through a porous cuticular plate at the
oral end of each frontal body. A groove in the epipharynx
connects each frontal body to the antero-dorsal region of the
bladder surface (1335).
Analyses of the frontal bodies and the fluid on the bladder
surface indicate that the total concentration of salts on the
bladder surface is less than 275 mEq, and the sum of the concentrations of organic solutes (sugars, amino acids, polyhydroxyl alcohols) is less than 2 mM. The fluid produced by the
frontal bodies is thus not hygroscopic and it must play a subordinate role in the WVA mechanism of Arenivaga (1330, 1336).
A speculative model for the mechanism of WVA in the
desert cockroach proposes that reduction of water activity
on the bladder surface is due to the properties of the fine
cuticular hairs, 0.1 mm in length and as fine as 180 nm in
diameter, which cover the bladders. Isolated samples of bladder cuticle were shown to be hydrophilic by measurement
of their water content over a range of humidities (1336). In
other words, the water content of bladder cuticle (g H2 O/g
dry weight) is much higher than that of unspecialized cuticle
from other regions of the body. Importantly, the water content of the hairs, measured gravimetrically or inferred from
changes in the volume of the hairs measured by scanning
electron microscopy, is highly sensitive to small changes in
ionic strength. Polyelectrolytes, such as chitin and protein in
the bladder cuticle, swell as the ionic strength of the surrounding medium is reduced (1804). Increases in salt concentration
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Osmoregulation and Excretion
reduce the mutual repulsive forces on a protein or gel structure and permit a greater number of non-covalent cross-links,
possibly of the van der Waals type, to form. As a consequence,
the protein or gel shrinks (888). Cyclical addition of frontal
body fluid may sufficiently alter ionic strength to promote
shrinkage of the bladder hairs, thereby releasing water from
the hairs. Some of this released water may evaporate, but
much will be swallowed by the substantial negative pressures
generated by the animal to pull fluid and condensate over the
bladder surface and into the esophagus. After the pulse or
wave of increased ionic strength passes over a particular area
of the bladder surface, the hydrophilic cuticular hairs will tend
to swell again by absorption of atmospheric water vapor. In
other words, condensation will dilute the surrounding fluid,
leading to further swelling, until swelling is constrained by
the elastic properties of the cuticular hairs (1333).
Soil-dwelling collembolans also provide an example of a
very different basis for water vapor absorption. Collembolans
are hexapods with internal mouthparts and are now considered
closely related to, but distinct from, the insects. Respiratory
gases are exchanged across the integument, whose permeability to water is high. They are thus in constant danger
of dessication and survive only hours at relative humidities
below 90%. However, they live in the pores between soil particles where the humidity is typically very close to 100% RH.
Accumulation of substantial quantities of sugars such as glucose, trehalose and polyols such as myo-inositol raises the
osmotic pressure of their body fluids. The consequent reduction in water vapor pressure allows passive absorption of water
vapor from the surrounding atmosphere at humidities above
95%, the lethal limit for drought tolerance in these animals.
In contrast to the insects, which are characterized by cuticle
with very low permeability to minimize water loss and in
which the mechanisms of water vapor absorption entail localized creation of extremely low water activity in specialized
tissues, collembolans have a highly permeable integument,
making localized active water absorption inappropriate. As
a result, these animals must maintain all of the body fluids
hyperosmotic to the surroundings to allow net water uptake
from the atmosphere by passive diffusion along the gradient
in water potential (793).
Agnatha and Pisces
All three possible strategies for maintaining salt and water
balance are represented by fish in marine environments.
(i) Osmoconformity/ionoconformity is found in the marine
agnathan hagfishes, which do not regulate osmotic pressure
and concentrations of main electrolytes to a great extent
(1251, 1604). (ii) Osmoconformity but regulation of main
ions is seen in marine and some euryhaline elasmobranch
(722) and in the lobe-finned coelacanth (649), which, in
marine environments maintains plasma osmolality slightly
above that of seawater but NaCl concentrations at 3035% of ambient levels. (iii) The most commonly observed
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Osmoregulation and Excretion
Comprehensive Physiology
strategy is osmoregulation, found in all teleosts (1167) and
lamprey (1251), which regulates main extracellular ions (Na+
and Cl− ) and osmotic pressure at approximately 150 mM
and 300 mOsm, respectively, regardless of ambient ion concentrations. Hagfish and lobe-finned fish are restricted to
marine environments. In freshwater, all fish regulate osmotic
Table 6
pressure and ionic concentrations above that of the environment. Examples of osmotic pressure, ionic and organic constituents of fluid compartments in fish are given in Table 6.
For a discussion of the evolutionary history leading to
the phylogenetic distribution of the above osmoregulatory
strategies, refer to Evans et al. (513).
Composition of Various Body Fluids from Fish
Hagfish
Environment
Elasmobranch
Marine
Freshwater
Marine
Fluid
Plasmaa
Urineb
Plasmac
Plasmad
Intestinal fluid -posteriord
Rectal gland secretionf
Urineb
Na+
549
533
178
263
356
540
240
Cl−
563
548
146
244
369
533
240
Urea
3
9
1
386
22
14.5
100
TMAO
–
–
–
36
6
–
10
Mg2+
19
15
–
1
30
<1
40
–
7
–
3
13
–
70
5
4
–
3
28
<1
3
11
11
–
5
8
7
2
–
–
–
4
1
–
pH
–
7.6
–
7.6
6.4
–
mOsm
–
–
320
947
950
1,018
SO4
2−
Ca2+
K+
HCO3
−
5.8
Teleost
Environment
Freshwater
Marine
Fluid
Plasmag
Intestinal fluidf
Bladder urineg
Plasmae
Intestinal fluid posteriore
Bladder urinee
Na+
139
130
4
166
28
34
Cl−
117
99
6
153
30
26
Urea
–
–
–
–
7
TMAO
–
–
–
–
–
Mg2+
1
1
1
153
95
SO4 2−
1
–
–
130
74
2
<1
2
5
–
Ca2+
3
K+
4
9
6
4
5
–
HCO3 −
20
–
5
53
–
pH
–
–
7.8
8.2
–
325
26
329
329
305
mOsm
290
Concentrations of urea, TMAO and electrolytes (mmol/kg or mmol/l) as well as pH and osmotic pressure in various body fluids from fish.
“–”; not determined or reported, a ; Atlantic hagfish (365), b ; Pacific hagfish and Spiny dogfish (749), c ; Potamotrygon sp. (2029), d ; Little skate
(34), e ; Gulf toadfish (1203), f ; freshwater-acclimated tilapia (653), g ; Northern pike (749), f ; Spiny dogfish (224).
446
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Agnata
Hagfish are found exclusively in the marine environment, and
their strategy for maintaining salt and water balance have been
discussed extensively recently (365, 509). Although hagfish
presumably do not need to drink at high rates to maintain water
balance, evidence for components of the renin-angiotensin
system (RAS) has been reported (324). It is possible that
the slightly lower osmotic pressure of the plasma compared
to seawater (Table 6) does require some seawater ingestion
for hagfish to maintain water balance. Although approximately isoosmotic to seawater, plasma K+ , Ca2+ , Mg2+ , Cl−
and SO4 2− concentrations are slightly below those of seawater, while Na+ concentrations seems to be slightly above
(Table 6). The transepithelial potential (TEP) across the hagfish integument remains to be reported, but regardless of what
it is, some of the aforementioned concentration differences
must reflect ions out of electrochemical equilibrium. While
extracellular fluids are similar to seawater, although not identical (see below), intracellular fluids contain lower concentrations of especially Na+ and Cl− with amino acids (2070 mmol/Kg) and trimethylamineoxide (TMAO) (∼210-230
mmol/Kg), but not urea, contributing to intracellular osmotic
pressure (86).
Gills
Although the hagfish gill morphology differs substantially
from that found in lamprey, elasmobranchs and teleost, (509)
the gill is still arranged to have counter-current blood and
water flow for efficient gas exchange and possesses numerous
mitochondria-rich (MR) cells. In addition to large amounts of
mitochondria, the cells are characterized by extensive basolateral membrane foldings that are continuous with an intracellular tubular system as seen in other fishes. However, deep
apical crypts, as seen in marine teleosts, are lacking (509). In
agreement with the limited need for branchial Na+ and Cl−
transport, Na+ /K+ -ATPase expression and enzymatic activity
levels are lower than commonly found in marine teleosts (288,
1146, 1862). Despite their limited branchial transport of Na+
and Cl− , hagfish are efficient regulators of acid-base balance
(476, 1861) via Na+ /H+ and Cl− /HCO3 − exchange systems,
suggesting that these systems originaly evovled for the purpose of acid-base balance regulation rather than osmoregulation (505). In agreement with these observations, carbonic
anhydrase, Na+ /H+ exchangers (NHEs) and the vacuolar H+ ATPase have been localized to hagfish branchial MR cells
(288, 291, 476, 1862), which likely serve acid-base balance
rather than osmoregulation.
Gastrointestinal tract
Recent studies have revealed modest drinking rates by hagfish held in normal seawater and a stimulated drinking rate
following abrupt transfer to 140% seawater (1812). Furthermore, increased concentrations of a non-absorbable marker,
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Osmoregulation and Excretion
polyethylene glycol, MW 4000 (PEG-4000), in intestinal fluids compared to seawater and a reduction in [NaCl] demonstrate the ability to perform solute coupled fluid absorption
across the intestinal tract (1812). However, solute coupled
fluid absorption by hagfish does not appear to be associated
with anion exchange and intestinal HCO3 − secretion, as is the
case for elasmobranchs and teleosts (below) (1812).
Kidneys
The hagfish kidney has 30-40 glomeruli that drain into paired
ducts, structurally similar to proximal tubules of other vertebrates (reviewed by 509). However, glomerular pressures are
so low that filtration may not occur (524, 1548), suggesting
that urine may be formed mainly by secretion (1553). Urineto-plasma ratios for Na+ , Cl− and inulin are approximately
1, indicating no water absorption and no secretion of monovalent ions by the renal tubules of hagfish (1265). In contrast,
Mg2+ and especially SO4 2− are concentrated in the urine
(Table 6) (1265), and secretion of these ions may thus drive
fluid secretion. A recent study reported that although hagfish
are incapable of regulating plasma osmotic pressure, divalent
ions appears to be potently regulated (1604), presumably by
renal excretion.
Elasmobranchs and Coelacanths
Little is known about osmoregulation by coelacanths, but their
blood is approximately isoosmotic with seawater, with ionic
composition very similar to the blood of elasmobranchs (649).
Coelacanths have what appears to be a salt secretion gland,
the postanal gland, which is similar to the rectal gland of elasmobranchs at the structural and ultrastructural levels (1032).
Furthermore, the postanal gland of coelacanths, as the rectal
gland of elasmobranchs, has high levels of Na+ /K+ -ATPase
activity (648) and is likely involved in secretion of Na+ and
Cl− . Given these similarities, the following discussion of salt
and water balance in elasmobranchs likely applies to coelacanths as well.
Marine elasmobranchs maintain plasma osmolality
slightly (∼20 mOsm) above ambient (790, 1714) and thus
gain osmotically “free” water and show very low, although
measurable, drinking rates (34, 40, 401). Plasma Na+ and
Cl− concentrations of marine elasmobranchs are maintained
much below those of the environment but higher than in
teleosts, with the main osmolyte being urea at concentrations
between 300 and 400 mOsm (Table 6). Urea appears to be
produced in the liver as well as the skeletal muscle (35, 876).
The denaturing effects of high osmotic pressure and
high urea concentrations are offset by high concentrations
of methylamines, especially trimethylamine oxide (TMAO).
Although subject to some controversy, the capacity for TMAO
production appears to be low and restricted to the liver, and
high TMAO levels are likely due mainly to effective retention (1856). Glomerular filtration rates and urine flow rates of
marine elasmobranchs are relatively high compared to marine
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Osmoregulation and Excretion
teleosts (512), and the urine is hypoosmotic, with Na+ and Cl−
being the main osmolytes. The kidney plays a central role in
osmoregulation by reabsorbing mainly urea and TMAO while
the rectal gland secretes the excess Na+ and Cl− load resulting from the inward-directed gradient of these ions. Little salt
excretion occurs at the gill, but the branchial epithelium is
nevertheless involved in osmoregulation by contributing to
urea retention despite substantial urea gradients between the
blood and water (see below).
Euryhaline and even freshwater elasmobranchs survive
in low salinity waters. Among euryhaline elasmobranchs,
the bull shark and the Atlantic stingray maintain elevated
plasma levels of urea even when in freshwater, although urea,
Na+ and Cl− levels are reduced resulting in lower osmotic
pressures compared to those observed in seawater-acclimated
animals (1452, 1458, 1459). Salt absorption by these two
species, when in low salinities, likely occurs via two distinct
populations of branchial MR cells. Uptake of Cl− appears
to take place via cells expressing the apical SLC26a4 anion
exchanger (Pendrin) and to be driven by basolateral V-type
proton ATPases, while Na+ uptake likely occurs via apical
NHE3 and is driven by basolateral Na+ /K+ -ATPase (1454,
1518).
At least two species of elasmobranchs, the Asian freshwater stingray and the stingray from Rio Negro, are capable of osmoregulation in freshwater. The Asian freshwater
stingray displays greatly reduced plasma urea levels (44 mM)
when in freshwater but maintains a fully functional ornithineurea cycle and is capable of increasing circulating levels
of urea in response to elevated ambient salinity (1803). In
contrast, the South American stingray features an inability
to retain urea when challenged with elevated salinity and
a degenerated rectal gland incapable of salt secretion (600,
1825, 1826, 1828). Although not conclusive, pharmacological evidence suggests that at least Na+ uptake in this species
occurs via ion exchange mechanisms (2029). For a review of
osmoregulation by euryhaline elasmobranchs, see Hazon et al.
(722).
Gills
The elasmobranch gill epithelium consists largely of pavement cells, although relatively large MR cells are present in
interlamellar regions and on the lamellae (289, 1858). Elasmobranch MR cells display microvilli on the apical membrane,
basolateral membrane infoldings, a tubulovesicular system in
the apical region and express Na+ /K+ -ATPase (509, 2005).
Despite the expression of Na+ /K+ -ATPase, elasmobranch gill
MR cells are likely not involved in NaCl secretion as evidence
for NKCC1, CFTR and K+ channels, which are all present in
the salt-secreting rectal gland (below), have yet to be reported.
Although not involved in NaCl secretion, MR cells of elasmobranch gills play a vital role in regulating acid-base balance
(1858, 1861, 1863) and contribute significantly to osmoregulation by regulating urea transport. Very low apparent urea
permeability of the gill is crucial for maintaining the high
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Comprehensive Physiology
plasma-to-water urea gradient (752, 1205, 1406) and is the
product of unique phospholipid bilayer composition as well
as specific “back transport” of urea from branchial epithelial
cells to the blood (531, 1406). The competitive inhibitor of
urea transporters, phloretin, increases urea efflux, presumably
by blocking a basolateral, Na+ -dependent urea transporter.
Transport by the basolateral Na+ -dependent urea transporter
is stimulated by ATP and is sensitive to ouabain, and thus
relies on Na+ gradients established by the Na+ /K+ -ATPase
(531).
Gastrointestinal tract
Contrary to common belief, elasmobranchs ingest seawater
(401, 1814), with drinking rates influenced by extracellular
fluid volume and controlled by the renin-angiotensin system
(33, 37-39, 41), as in teleosts (below). Very little is known
about ingested seawater processing by the elasmobranch gastrointestinal tract, but they are capable of performing solutecoupled fluid absorption across various segments of the intestine (34, 40, 1048, 1814). Absorption of Na+ and Cl− appears
to drive water absorption, and substantial intestinal HCO3 −
secretion rates suggest that part of the Cl− absorption occurs
via anion exchange as is the case for marine teleosts. However,
nothing is known about the nature of the anion exchange proteins and the possible involvement of Na+ :Cl− cotransporters
as seen for marine teleosts (below). The main osmolyte in
marine elasmobranchs, urea, appears to be lost across the
intestinal epithelium as evident from luminal concentrations
as high as 375 mM in the proximal segments of the intestine (34). Interestingly, the majority of this urea is reabsorbed
by the distal intestinal segments as the concentration falls to
<22 mM. Although the mechanism of this urea reabsorption
remains to be elucidated, high expression of a urea transporter and a member of the Rhesus-like ammonia transporters
(Rhbg) suggest that these transporters might be involved in
reclaiming the lost nitrogen (34). Considering that many elasmobranchs are intermittent feeders, nitrogen limitations have
been proposed for this group of vertebrates (2023, 2024, 2028,
2030). Indeed, elasmobranchs examined so far show a remarkable ability to retain nitrogen even after feeding (2028), at a
time when teleost fish show high rates of ammonia excretion
(1816).
Kidney
Tubule anatomy The complexity of the elasmobranch
kidney rivals that of mammals, as reviewed recently (509).
The nephrons are glomerular followed by five segments generally termed the neck, the proximal, intermediate and distal
segments, ultimately leading to the collecting tubules and the
collecting duct. From a functional perspective, it is important
to observe that these five segments are arranged in four loops
such that tubular fluids travel in opposite directions in five
closely adjacent tubular segments from the same nephron,
forming a renal counter-current system (165, 988).
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Comprehensive Physiology
Glomerular filtration and urine flow rates Glomerular filtration rates average 4 ml/kg/h in marine elasmobranchs
(749) and appear to be regulated by the number of filtering nephrons (910, 1655, 1723), as is the case for marine
teleosts (below). Urine flow rates are substantially lower than
glomerular filtration rates are, demonstrating 60%-85% tubular fluid absorption (749, 910, 1723), rates closely associated
with absorption of urea and TMAO. Although secretion may
occur in the proximal tubules (120), filtration and reabsorption
are the important renal processes for marine elasmobranch
osmoregulation (115), and elasmobranch urine is typically
hypoosmotic by 50-250 mOsm (222).
Tubular secretion and reabsorption Secretion by the
elasmobranch proximal tubule appears to be similar to the
active extrusion of monovalent ions, driving fluid secretion,
observed in teleost proximal tubules (below) (115, 1612). In
brief, transcellular Cl− secretion is achieved by a basolateral NKCC1 and an apical CFTR-like channel while Na+
follows via a paracellular shunt. Na+ /K+ -ATPase provides
the gradients for operation of basolateral NKCC1, with K+
recycling across the basolateral membrane being facilitated
by K+ -channels (120, 1612). This NaCl secretion mechanism is identical to those observed in the elasmobranch rectal
gland (below) and the marine teleost gill (below and Fig.
18). Both Mg2+ and SO4 2− concentrations in the urine are
well above what could be accounted for by fluid absorption
(Table 6), suggesting tubular secretion as in teleosts, but nothing is known about elasmobranch tubular secretion of these
divalent ions.
The most efficient absorption by the elasmobranch
nephrons is that of urea and TMAO, with more than 90%
of the filtered load being absorbed (543). Although urine
Na+ and Cl− concentrations are similar to plasma concentrations (Table 6), substantial absorption occurs and likely
contributes to fluid absorption. Studies on the isolated third
loop of the nephron (late proximal and intermediate segment)
demonstrated Cl− absorption via NKCC2 (559), which was
supported by observations of NKCC mRNA in elasmobranch
nephrons with apical membrane localization of the gene product (131, 2047). Urea reabsorption has long been recognized
to be linked to Na+ absorption in a fixed ratio over a wide
range of urine flow rates (1630), and can, in part, be accounted
for by solvent drag as ∼70% of the filtered fluids are being
reabsorbed. However, since urine urea concentrations are well
below those of the plasma, excess urea reabsorption must
occur. Based on the above observations, it has been proposed
that NaCl absorption from the lumen of loop 3 tubules produces hypertonicity in the surrounding extracellular fluids,
which in turn creates an osmotic gradient for fluid movement from the lumen of the adjacent loop 4; the resulting
increased concentration of tubular urea favors urea movement
from the lumen of the collecting tubules to the surrounding tissues (559). Carrier-mediated urea transport by elasmobranch renal tubules has long been assumed (718, 910, 988,
Volume 4, April 2014
Osmoregulation and Excretion
1626, 1630) but is now conclusively demonstrated. A facilitated urea transporter (ShUT) cloned from the spiny dogfish
exhibits robust renal expression and phloretin-sensitive urea
transport when expressed in Xenopus oocytes (1712) and has
since be reported from other elasmobranchs (820, 840). ShUT
is found exclusively in the final segments of the tubule (820),
which is in agreement with the proposed role for this segment
in urea absorption. More recently, two distinct apical urea
transporters displaying non-saturable phloretin sensitive urea
transport (ShUT) and saturable, phloretin sensitive Na+ :urea
cotransport have been characterized from elasmobranch kidneys (1249). Interestingly, both these transporters support the
links between Na+ and urea absorption, since ShUT relies
on urea gradients established by NaCl and water absorption while the Na+ :urea cotransporter relies directly on Na+
transport.
Rectal gland
The function of the spiny dogfish rectal gland in salt homeostasis was first demonstrated in 1960; this organ secretes
isoosmotic fluid comprised almost exclusively of Na+ and
Cl− (Table 6) (224). The concentrations of these monovalent ions are approximately double those of the plasma, and
the gland therefore contributes substantially to Na+ and Cl−
elimination. Although the spiny dogfish can compensate for
rectal gland removal by elevated renal salt clearance, the lack
of a rectal gland compromises the ability to clear injected
NaCl from the plasma (223). Similar findings have since been
reported for other elasmobranchs (269, 719).
The rectal gland drains into the distal intestine and secretions exit via the cloaca. The gland is supplied by simple vasculature and secretions are carried by a single duct, making
this organ well suited for perfusion experiments on isolated
preparations or in situ (1693, 1728, 1729). The ultrastructure of the rectal gland has been reviewed comprehensively
(1369).
The molecular pathways mediating Na+ and Cl− secretion are well known, were summarized recently (509) and
are thought to be identical to those described for Na+ and
Cl− secretion by the marine teleost gill (below and Fig. 18).
Hypervolemia stimulates rectal gland secretion via secretion
of cardiac natriuretic peptide (1636, 1728, 1729), which is
potentiated by local release of vasoactive intestinal peptide
from rectal gland nerves (1692). Cardiac natriuretic peptide acts via the guanylate cyclase, protein kinase pathway
(680, 1691), while vasointestinal peptide acts via cyclic AMP
(312, 722). In addition, gland secretions appear to be correlated with blood perfusion, which is limited under physiological conditions by catecholamine induced vasoconstriction
(1682, 1683). Finally, stimulation of the extracellular calciumsensing receptor CaR causes gland vasoconstriction. Elevated
plasma Na+ and Cl− concentrations act to impair CaR activation in the presence of constant Ca2+ concentrations, and thus
release vasoconstriction for increased blood flow and thereby
gland secretion (522, 523).
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Osmoregulation and Excretion
Freshwater Fish
Overview
The following likely apply to freshwater lamprey, elasmobranchs and teleosts, although studies on teleosts are by far
the most abundant. Gas exchange by fish is achieved by a large
branchial surface area and intimate contact between the blood
and the surrounding medium. However, these properties of the
gill that allow for sufficient gas exchange despite low oxygen
solubility in water, combined with large osmotic gradients
between extracellular fluids and the freshwater environment,
result in diffusive water gain. Consequently, freshwater fish
produce large volumes of dilute urine (see Table 6 for urine
composition). Although the renal tubules in freshwater fish
are specialized for electrolyte absorption, renal ion loss is
unavoidable, adds to diffusive ion loss across the gills and
must be compensated by ion uptake. Ions are gained from the
diet and by active uptake across the gill epithelium mainly by
specialized MR cells.
Absolute ion uptake rates are variable among freshwater organisms, including fish, but exhibit a strong dependence of size (664), presumably due to size-dependent massspecific metabolic rate and relative gill surface area; the
greater the mass-specific metabolic rate, the greater the relative gill surface area and thus diffusive ion loss to be compensated by active ion uptake. An analysis, including 50 studies of Na+ uptake by freshwater organisms, revealed that
65% of the overall variation can be attributed to size (LOG
uptake = 2.87 − LOG(Mass)·0.274, where uptake rates and
mass are in nmol/gram/hour and grams, respectively (664).
According to this relationship a standard 1 gram fish displays Na+ uptake rates of ∼750 nmol/gram/hour, while a
standard 100 gram fish takes up Na+ at a rate of ∼ 210
nmol/gram/hour to maintain homeostasis. Other factors obviously play a role in dictating Na+ uptake rates, among
which are ambient Na+ concentrations. Affinity and maximal transport capacity of the Na+ (and Cl− ) uptake pathways of freshwater fish range from 13 μM to 18,500 μM
and from 380 nmol/gram/hour to 19,000 nmol/gram/hour,
respectively (150, 200, 507, 553, 1143, 1408, 1481). Both
parameters are sensitive to ambient ion concentrations and
are adjusted as fish acclimate to different ion concentrations with both the uptake affinity and maximal transport
capacity increasing as ambient ion concentrations decrease
(150, 200).
Gills
Ion transport proteins are detected in MR cells and pavements
cells of freshwater fish gills (1168, 2004), suggesting that
both cell types may contribute to ion transport across the gill
epithelium. However, because pavement cells lack substantial metabolic potential to drive transport, the less abundant
MR cells likely contribute most to ion uptake. Indeed, strong
correlations have been observed between the relative MR cell
exposed apical surface area and Na+ uptake rates in several
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species of freshwater fish (200, 1427), strongly supporting
this notion.
The morphology (and function) of fish gill MR cells has
been reviewed most recently in 2005 (513). Morphologically,
electron-dense β-cells and less dense α-cells have been identified in gills of freshwater teleost fish or freshwater-acclimated
euryhaline teleost fish (1463, 1464, 1466, 1467). Based on
morphological observations on gills obtained from fish in different environments, α-cells were suggested to conduct Cl−
secretion in seawater and Na+ uptake in freshwater, while
β-cells were proposed to be associated with Ca2+ uptake in
freshwater (1462, 1465). Similarly, more than one MR cell
type is present in the gills of freshwater-acclimated lamprey
(76, 77, 1252, 1291, 1413).
Na+ uptake models No less than three models for Na+
uptake by freshwater fish have been proposed.
(i) The classic model of Na+ /H+ (NH4 + ) exchange, first
proposed by Krogh (973) and since attributed to apical isoforms (NHE2 and NHE3) of the SLC9A gene
family, driven by electrochemical Na+ gradients established by the basolateral Na+ /K+ -ATPase, have been
challenged recently due to apparent thermodynamic constraints on Na+ uptake via this pathway (55, 1403). Nevertheless, occurrence of branchial NHE2 and NHE3 in a
high number of species including agnathans (291, 476),
elasmobranchs (291, 477, 1858) and teleosts (263, 300,
771, 2004) has been demonstrated. Furthermore, transfer of euryhaline species to lower salinities results in
increased expression of NHE2 (Fundulus heteroclitus;
[1642]) and NHE3 (Dasyatis sabina; [290]), transfer
of zebrafish to low Na+ -freshwater causes an increased
branchial expression of NHE3b (2049), and hypercapnic acidosis stimulates expression of NHE2 in freshwater acclimated rainbow trout (835). In addition, the
amiloride derivate, EIPA, which is assumed to be a specific inhibitor of NHEs also in fish (771), inhibits Na+
uptake in freshwater-acclimated Cyprinodon variegatus variegatus and Cyprinodon variegatus hubbsi (200).
These observations suggests that electroneutral Na+ /H+
exchange may occur via NHE members of the SLC9A
family despite apparently unfavorable conditions. It is
possible that activity of the Na+ /K+ -ATPase in the extensively folded basolateral membrane, which creates a
tubular system in close proximity to the apical membrane
(1445), establish low Na+ -microenvironments allowing
for the apical NHEs to function even in freshwater environments (771). Recent studies on zebrafish and medaka
link the apical rhesus protein Rhcg1 to the NHE3b isoform in a way where NH3 appears to be excreted via
Rhcg1 and be trapped by conversion to NH4 + with protons provided by NHE3b (980, 1666, 2043). In turn,
the removal of protons from the apical boundary layer
by conversion of NH3 to NH4 + facilitates the continued
operation of NHE3b allowing for Na+ uptake. While
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Comprehensive Physiology
these studies provide additional support for the Na+ /H+
exchange model, it should be noted that it remains to be
seen if the Rhcg1/NHE3 constellation applies generally
to freshwater fish.
(ii) An alternative model for Na+ uptake was proposed based
on findings of the vacuolar H+ -ATPase in the apical
region of rainbow trout gills (1058-1060) and was later
confirmed for several freshwater species by observations of reduced or abolished Na+ uptake in the presence of the specific inhibitor bafilomycin (1-2·10−6 M)
(150, 526, 666), although some species clearly lack an
apical H+ -pump (200, 1408). Localization of the H+ pump in the apical region has also been demonstrated for
zebrafish (150) and is assumed to fuel Na+ uptake via
Na+ channels by hyperpolarization of the apical membrane. Searches for an obvious Na+ channel candidate
involved in freshwater fish Na+ uptake, the epithelial
Na+ channel (ENaC), have so far been fruitless, and
ENaC is not present in the teleost genomes sequenced
to date, leading to the conclusion that other Na+ (or
unspecific cation) channels are involved in Na+ uptake.
Regardless of the nature of the Na+ channel involved,
it is sensitive to the amiloride derivate, phenamil, at
least in some species (229, 664), and appears to be
subject to rapid, non-genomic downregulation by elevated ambient Na+ concentrations (150) much like ENaC
in isolated frog skin (562, 589). Interestingly, acclimation to low ambient Na+ fails to induce increased
expression of the H+ pump, although acidic environments potently induce H+ pump expression in zebrafish
(ZF). These observations suggest that the Na+ channel
rather than the H+ pump is limiting for Na+ uptake
and illustrates the dual role of the branchial H+ pump
in osmoregulation and acid-base balance in freshwater
fish.
(iii) A third proposed model for Na+ (and Cl− ) uptake
involves absorptive Na+ -2Cl− -K+ (NKCC2) or Na+ :Cl−
cotransporters (NCC). Regardless of which of these
cotransporters are considered, thermodynamic considerations are difficult to reconcile with Na+ (and Cl− )
uptake in freshwater. Nevertheless, the evidence for their
presence and contribution to ion uptake is growing. Early
observations of bumetanide-sensitive ion uptake by goldfish (1481) have since been supported by immunohistochemical observations of branchial NKCC isoforms in
the apical region for freshwater-acclimated fish (772,
773, 829). More recently, NCC was identified in MR
cells from zebrafish and tilapia (818, 819) and localized to the apical membrane of certain MR cells in
zebrafish. Furthermore, knockdown experiments with
zebrafish embryos demonstrated a role for this transporter in Cl− uptake (1930) while the use of the dye,
Na+ green, implicated NCC in Na+ uptake by zebrafish
as well (502).
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Osmoregulation and Excretion
The H+ pump undoubtedly contributes to Na+ uptake
across the apical membrane in some species, possibly depending on ambient NaCl concentrations, and basolateral Na+ /K+ ATPase (NKA) clearly is important for transepithelial Na+
transport by maintaining low cellular Na+ concentrations and
directly extruding Na+ across the basolateral membrane. The
NKA is highly expressed in gill epithelia (771, 1051, 1201,
1417, 1905, 2004) and localizes to the basolateral membrane
or the tubular system of the MR cells (373, 374, 1451), but
demonstrates differential subunit composition dependent on
ambient salinity. The α1a and α1b subunit isoforms in the
gills of rainbow trout are expressed reciprocally and have
been proposed to play a role in Na+ uptake in freshwater and
Na+ extrusion in seawater, respectively (1540); the α1a isoform is upregulated in a number of salmonids when acclimating to freshwater (238, 239, 1680, 1835). Three amino acid
substitutions in transmembrane segment 5 (TM5), TM8 and
TM9 separate the α1a isoform from the α1b (857). Together
these three amino acid substitutions promote binding of Na+
over K+ from the cytoplasm, reduce the Na+ /ATP ratio and
the work performed on a single pump cycle and furthermore,
inhibit binding of K+ such that Na+ /H+ rather than Na+ /K+
exchange is promoted (857). These changes in the α1a subunit seem to be highly adaptive for osmoregulation in low ion
environments by facilitating Na+ uptake against very steep
gradients (10-20 μM in water versus 150 mM in blood).
Interestingly, a number of α1 subunits have also been identified in zebrafish and cichlids and show differential expression depending on ambient water ion concentrations (1046,
1833). Although the functional differences of the α1 subunits
in zebrafish remain to be fully determined, these observations suggests that mechanisms similar to those discussed for
salmonids above may also be present in cyprinids. Furthermore, FXYD proteins co-localize with NKA, are differentially
expressed depending on water chemistry and “seawater readiness” in salmonids, suggesting a regulatory role of FXYD on
NKA activity (1831, 1837).
Chloride uptake Salmonids (633, 635), cyprinids (150,
526, 977) and cichlids (271, 526) rely on active uptake of
Cl− across the gill epithelium while certain anguillids (153,
633, 659, 929) and centracids (1841) display no Cl− uptake
from freshwater. As discussed above, Na+ :Cl− cotransport
systems may contribute to Cl− uptake by certain freshwater
species. However, early observations by Krogh provided evidence for Cl− uptake via Cl− /HCO3 − exchange (973, 977),
an observation that has since been confirmed. Induction of
metabolic alkalosis causes an increase in HCO3 − excretion,
Cl− uptake and an increase in MR cell surface area in rainbow
trout, providing direct evidence for Cl− /HCO3 − exchangers
and their localization to MR cells in the gill epithelium (634,
1426). Two groups of Cl− /HCO3 − exchangers are found in the
SLC4 (HCO3 − transporters) and SLC26 (anion transporters)
gene families (18, 1258, 1572). Results of in situ hybridization (1788) and immunohistochemistry (2004) experiments
implicate SLC4a1 (AE1) in Cl− uptake, although these
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Osmoregulation and Excretion
observations should be interpreted with caution because heterologous probes and antibodies were used. More recently,
SLC26a4 (Pendrin) was identified in freshwater acclimated
stingray gills (1454), and SLC26a3, SLC26a4 and SLC26a6
were all identified in developing zebrafish embryos (81).
Furthermore, these transporters are regulated at the transcriptional level depending on ambient Cl− or HCO3 − , and
gene knockdown experiments (morpholinos) demonstrated
reduced Cl− uptake when transcription of SLC26a3, a4 and
a6 was blocked (81). Similar experiments on adult zebrafish
confirmed that mRNA expression of SLC26 transporters
responds to lowered Cl− and elevated HCO3 − concentrations in water and shows apparent MR cell localization, and
that induced changes in expression mirror absolute and coupled Cl− uptake and HCO3 − excretion rates (1430). While
the evidence for SLC26 anion exchangers in freshwater fish
Cl− uptake is strong, it is not entirely clear how electroneutral anion exchange can overcome rather unfavorable gradients for Cl− uptake; freshwater fish are capable of taking
up Cl− from less than 10 μM (150). However, apical proton pump activity appears to drive apical anion exchange,
since bafilomycin inhibits Cl− uptake, by allowing for cellular accumulation of HCO3 − from CO2 hydration and possibly
by maintaining low HCO3 − concentrations on the unstirred
boundary layer at the apical surface. The observation of electrogenic SLC26a6 operating in an nHCO3 − /Cl− mode in
marine teleosts (662, 982) offers an interesting perspective
for Cl− uptake by freshwater organisms since such a stoichiometry would benefit from the apical membrane potential and the activity of the electrogenic H+ -pump, and as
such would permit Cl− uptake from freshwater even at low
ambient Cl− concentrations (662). To date, the stoichiometry of freshwater fish SLC26 anion exchangers has not
been examined. Exit of Cl− across the basolateral membrane is believed to occur via a CFTR-like Cl− channel (1166,
1168, 1696).
Cellular substrate for ion exchange—carbonic anhydrase Aside from NaCl uptake via cotransporters, uptake
of both Na+ and Cl− requires cellular substrate in the form
of H+ and HCO3 − , respectively (636). Intracellular carbonic
anhydrase (CAII-like, termed CAc in rainbow trout; [599]) is
present in MR cells as well as pavement cells (599, 615, 771,
1064, 1497) and plays a role in Na+ and Cl− uptake (150,
912, 1429) by facilitating hydration of CO2 to produce H+
and HCO3 − .
MR cell diversity
The morphological subtypes of MR cells first described by
Pisam and coworkers (1463, 1464, 1466) show strong resemblance to two subtypes of intercalated cells (α-cells and
β-cells) in the mammalian kidney. Peanut lectin agglutinin
(PNA) was found to only bind to the β-subtype (β-type)
of intercalated cells and has been used successfully to differentiate between these two mammalian kidney cell types
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Comprehensive Physiology
(1030). More recently, PNA binding has been employed to
differentiate and isolate two types of MR cells (PNA− and
PNA+ , corresponding to α-types and β-types, respectively)
from freshwater-acclimated rainbow trout gills (576, 632) and
has paved the way for partial physiological characterization
of these two cell types (576, 1400, 1402) (Fig. 17). Since
these pioneering studies on freshwater rainbow trout, MR
cell diversity has been documented from zebrafish and tilapia
as recently reviewed (818) (Fig. 17). At least three subtypes
of MR cells have been identified in zebrafish: vacuolar H+ pump rich (HR) cells, Na+ /K+ -ATPase rich (NaR) cells and
Na+ :Cl− cotransporter (NCC) cells (Fig. 17). All three cell
types show strong expression of Na+ /K+ -ATPase (818). HR
cells are acid-secreting cells displaying Na+ uptake (501,
800, 801, 1062). Scanning ion-selective electrode techniques
(SIET) where employed to demonstrate bafilomycin-sensitive
H+ currents in cells expressing V-type ATPase mRNA and
protein (1062). Translational knockdown (morpholinos) of
the H+ pump was found to decrease Na+ concentrations in
HR cells and whole-body zebrafish, providing evidence for a
role in Na+ uptake (501, 800). As discussed above, the quest
for an ENaC-like Na+ channel in fish has been fruitless so far,
and indeed, pharmacological and molecular evidence support
the involvement of NHEs in zebrafish Na+ uptake (150, 501).
Specifically, NHE3b mRNA is expressed predominantly on
HR cells and is upregulated following acclimation to low
Na+ water, implicating this transporter in Na+ uptake by HR
cells (2049). Interestingly, NHE2 and NHE3 have been documented to localize to trout PNA-positive cells (835). The
presence of NHE3b and the H+ pump in the same cell type
may seem counterintuitive, but it appears from manipulations of ambient pH and Na+ concentrations that NHE3b
is primarily regulated based on Na+ availability while the
H+ pump is regulated by ambient pH (2049) suggesting distinct roles for these two transporters. The anion exchangers
(SLC26a3, a4 and a6) recently implicated in Cl− uptake by
zebrafish (81, 1430) have yet to be localized to specific MR
cells. However, the apparent dependence on H+ extrusion
(150) points to a role for HR cells in Cl− uptake via anion
exchange.
Not surprisingly, HR cells express an apical, extracellular
membrane-associated carbonic anhydrase (CA)15a isoform
and cytosolic CA2 (equivalent to trout CAc), and pharmacological manipulations as well as morpholino experiments have
demonstrated a role for acid excretion as well as Na+ uptake
(150, 501, 1064). In agreement with these observations, trout
CAc has been demonstrated to localize to MR cells in trout
gills and to play a role in acid-base regulation (599). NaR
cells show strong expression of Na+ /K+ -ATPase but appear
to be primarily involved in Ca2+ uptake via apical epithelial
calcium channels (ECaC), basolateral Ca2+ ATPase and the
Na+ /Ca2+ exchanger (NCX) (1047, 1396). The activity of the
Na+ /K+ -ATPase likely provides conditions for the operation
of NCX. NCC cells express a Na+ :Cl− cotransporter isoform,
and loss-of-function experiments (morpholinos) showed
significant decreases in Cl− uptake and Cl− content of embryo
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Comprehensive Physiology
Osmoregulation and Excretion
Rainbow trout
PNA+ cell
Na+
NHE2/3
+
H
K+
~
Na+
NCC cell
NCC
Cl–
Tilapia
Na+
K+
Cl–
SLC4a4
~
~
Na+
Type 1
K+
Na+
~
Na+
HCO3–
PNA– cell
K+
~
Na+
NaR cell
K+
~
Na+
Type 2
K+
Na+
Na+
Cl–
HCO3–
~
Na+
SLC26a3, a4, a6
HCO3–
H+
Zebrafish
NCC
Na+
SLC4a4
–
Cl
HCO3–
SLC26a3, a4, a6
H+
HR cell
~
K+
Na+
H+
Water
CA15
HCO3–
–
Na+
Type 3
Na+
H+
CA2
NHE3
~
Cl
SLC26a3, a4, a6
Cl–
NHE3
K+
Na+,K+,2Cl–
SLC4a1
HCO3–
Type 4
K+
–
Cl
~
Na+
NKCC
~
Na+
CFTR
Na+,K+,2Cl–
NKCC
Figure 17
Schematic representation of accepted (black) and putative (gray) transporters and processes in freshwater fish gill MR cell types
(modified from [818]). Classification of cell types is based in part on immunohistochemistry and in part of transport properties of individual cells as
assessed by ion-selective electrodes or isolation of individual cell types. All MR cells express high levels of basolateral Na+ /K+ -ATPase. Rainbow
trout PNA+ cells express apical NHE isoforms while the PNA− cells express apical H+ -pumps and a Na+ -channel yet to be identified. Both
PNA+ and PNA− cells take up Cl− via Cl− /HCO3 − exchange processes (1402), and although the nature of the anion exchanger(s) involved are
unknown, members of the SLC26 family are likely candidates. Zebrafish possess at least three distinct MR cell types. NCC cells are characterized
by apical an Na+ :Cl− cotransporter (NCC) and a basolateral Na+ :HCO3 − cotransporter (SLC4a4) (1028). The NaR cell type, in addition to
high levels of Na+ /K+ -ATPase, contains apical and basolateral Ca2+ transporters (not shown) and is likely involved in Ca2+ acquisition. The
third zebrafish cell type, HR, shows strong expression of apical H+ -pumps and expression of apical NHE3 as well as basolateral SLC4a1 (1028).
Furthermore, cytosolic carbonic anhydrase (CA2) and membrane bound extracellular carbonic anhydrase (CA15) in HR cells are involved in ion
uptake. Uptake of Cl− by zebrafish occurs via three members of the SLC26 gene family which have yet to be localized to specific cell types.
However, due to the dependence of Cl− uptake on H+ -pump activity, the SLC26 transporters have been indicated in NR cells in the present
representation. The euryhaline tilapia acclimated to freshwater have as many as four unique MR cell types. So far, type 1 cells have only been
demonstrated to express basolateral NKA, and their function remains to be elucidated. Tilapia cell type 2 appears identical to zebrafish NCC
cells, with apical NCC and basolateral SLC4a4 cotransport, while cell type 3 express apical NHE3 and basolateral NKCC (presumably NKCC1).
Basolateral NKCC(1) is also found in tilapia cell type 4, which also express apical CFTR Cl− channels. It should be noted that the representations
illustrate what currently is supported by the peer-reviewed literature, and that multiple additional transporters and enzyme not included in these
diagrams must also be present to account for the observed transport properties of the freshwater teleosts fish gill.
morphants (1930). Multiple types of MR cell are seen also
in tilapia embryonic skin, which display types I, II, III and
IV (Fig. 17), classified by differential labeling for different
transporters (773, 829). All four subtypes display strong basolateral staining for Na+ /K+ -ATPase. To date, no other transporters have been identified in Type I cells. Type II cells show
apical NCC localization and are analogous to the zebrafish
NCC cells (818), while Type III cells show basolateral NKCC
and apical NHE3 staining and seems analogous to HA cells
from zebrafish. Type IV cells from tilapia held in freshwater appears to be a seawater-type MR cell (see below) with
Volume 4, April 2014
basolateral NKCC and apical CFTR staining. Experiments
allowing fish to acclimate to distinct water chemistries suggested that Type II cells are involved in Na+ and Cl− uptake
and that type III cells are contributing to Na+ uptake, while
Type IV is responsible for Na+ and Cl− secretion (773, 829,
830).
Gill epithelial permeability
Much focus has been placed on the active Na+ and Cl− uptake
pathways in freshwater fish, with less attention directed
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Osmoregulation and Excretion
toward potential control of the passive branchial efflux component. A high number of studies have demonstrated substantial differences in trans-gill epithelial potential (TEP) among
euryhaline fish, depending on exposure to different ambient
salinities, and furthermore, that these differences are attenuated by salinity acclimation (reviewed by [1167]). More
recently, a detailed time course revealed that TEP changes
gradually over a 12-30-hr period after transfer of seawater acclimated killifish (Fundulus heteroclitus) to freshwater,
settling at levels representative of fully freshwater acclimated
individuals (2025, 2027). These observations were interpreted
as changes in the relative permeability of Cl− compared to
Na+ (PCl /PNa ), probably associated with closing of the paracellular Na+ shunt pathway (2025). Indeed, relatively rapid
reductions in Na+ efflux have been reported for fish exposed to
reduced ambient Na+ concentrations (200, 695), while a single study has demonstrated similar responses for Cl− (1428).
Changes in gill permeability are thought to be related, in part,
to altered lipid bilayer composition (695, 696), and more
recently the tight junction proteins, occludin and claudins,
have been implicated in the control of gill epithelial permeability. Occludin is a four-transmembrane protein exhibiting
a physiological role in determining the barrier properties of
vertebrate epithelia (521, 624), and recently documented to be
present in fish epithelia, including the gill (274-277). Occludin
expression was elevated in goldfish following transfer to ionpoor water (274), and occludin protein levels in rainbow trout
gill epithelial cultures are inversely correlated with epithelial permeability (277). These observations point to a role for
occludin in controlling epithelial barrier functions in freshwater fish gills.
Like occludin, claudins are four-transmembrane domain
proteins and integral components of tight junctions (1879).
The physiology of claudins is an emerging field complicated
by their diversity; at least 24 claudins are known from mammals and no less than 56 are found in the puffer fish, Fugu
rubripes (1091, 1901). Nevertheless, evidence so far suggests
that a number of claudins (claudins 3, 3a, 3c, 8d, 27a, 27b,
28a, 30) may be upregulated in gills of euryhaline fish (tilapia,
salmon, puffer fish and eels) acclimated to freshwater, where
they play a role in tightening the branchial epithelium (58, 59,
459, 879, 1832, 1834, 1835).
Gastrointestinal tract
By far the majority of studies of osmoregulation by freshwater
fish have been performed on fasted animals to avoid fouling
of the water in closed systems most commonly used for ion
uptake measurements. Consequently, little is known about
the dietary contribution to osmoregulation by freshwater fish.
However, the question of potential dietary contributions to salt
homeostasis in freshwater fish has received some attention in
the past few years (216, 217, 2024); the field has been summarized and synthesized recently (2023). Natural diets for
fish, consisting of aquatic invertebrates, contain 34-41 mmol
Na+ /Kg (947). Assuming a food consumption rate of 3%
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Comprehensive Physiology
body mass per day and complete assimilation of dietary Na+ ,
dietary intake would amount to 20-24% of the branchial Na+
uptake rates of a standard 100-gram fish. Consistent with this
relatively modest predicted contribution are observations of
no net Na+ gain from ingested diets in freshwater acclimated
rainbow trout (216). However, it should be noted that dietary
Na+ uptake may play a more significant role in freshwaters
of low ionic strength where branchial uptake maybe limited.
For example, at ambient Na+ concentrations, branchial Na+
uptake by the freshwater stingray (Potamotrygon sp.) is insufficient to offset the diffusive Na+ loss (2029). The obvious
conclusion is that dietary Na+ uptake is of quantitative significance for salt balance in this freshwater elasmobranch.
In contrast to the low Na+ absorption observed in rainbow
trout, 89% and 81% of ingested K+ and Cl− , respectively,
was assimilated by the same fish suggesting that dietary Cl−
and especially K+ may serve an important source of these
electrolytes to freshwater fish (216). The pathway for dietary
K+ uptake in freshwater fish is unknown, but Cl− uptake from
the diet appears to occur in exchange for HCO3 − secretion
(215, 338, 1816), which acts to neutralize acidic chyme from
the stomach and alleviate, or at least reduce, the alkaline tide
in fish compared to that seen in many postprandial terrestrial
vertebrates (1321).
Kidney and urinary bladder
Due to the diffusive water gain mainly across the gill surface,
urine flow rates are generally high in freshwater fish ranging
between 2 ml/kg/h and 10 ml/kg/h (193, 366, 668, 749, 1207).
Tubular reabsorption of filtered ions is efficient such that the
dominant electrolytes, Na+ and Cl− , are typically less that
5-10 mM with other electrolytes being in the submillimolar
range (749). Thus, net renal Na+ loss is in the range of 10-100
nmol/gram/hour, which has led to the common perception that
renal NaCl handling is of minor significance for osmoregulation. However, considering a glomerular filtration rate of
5 ml/kg/hour and a plasma Na+ concentration of 150 mM,
renal Na+ filtration occurs at a rate of 750 nmol/gram/hour.
More than 90% (668), or 675 nmol/gram/hour, of the filtered
Na+ is reabsorbed by the renal tubules, which compares to
branchial Na+ uptake rates of 210 nmol/gram/hour in a standard 100-gram fish. Thus, renal transport processes are highly
significant for freshwater fish volume control as well as ion
regulation.
Tubule anatomy In contrast to marine conspecifics, freshwater fish kidneys have distal renal tubules connecting proximal segments to the collecting duct via a narrow, short intermediate segment and collecting tubules (749). With very few
exceptions, freshwater fish have glomeruli contained in renal
corpsules (1167). At least three evolutionary events have led
to the loss of glomeruli, presumably in species inhabiting
marine environments, but aglomerular fish are found in freshwater (115, 329, 330). Obviously, urine production in these
Volume 4, April 2014
Comprehensive Physiology
species occurs strictly by secretion as discussed for marine
teleost kidneys below.
Glomerular filtration and urine flow Urine formation
in freshwater fish consists of glomerular ultrafiltration and
substantial reabsorption of mainly monovalent ions across
the tubular epithelia. The tubular epithelium of freshwater
teleost kidneys exhibits low water permeability, with the lowest permeability seen in the most distal regions. However,
some water is absorbed with the reabsorption of electrolytes
and organic solutes such that urine flow rates generally are
lower than glomerular filtration rates (749). In contrast to the
situation for terrestrial vertebrates that display regulated tubular water reabsorption, urine flow rates in freshwater teleosts
are directly proportional to glomerular filtration regardless of
metabolic rate (748, 749, 784). In contrast to mammals, fish
display glomerular intermittency, and whole kidney glomerular filtration is the product of all-or-none regulation of individual nephrons (193, 208, 749).
Tubular reabsorption
As in mammals, some reabsorption of electrolytes occurs
in the proximal tubules, which are also the site for glucose
and HCO3 − reabsorption (218, 598). However, the majority of ion absorption by freshwater fish tubules occurs in
the water-impermeable distal tubules (193). In the proximal tubule, basolateral Na+ /K+ -ATPase drives Na+ uptake
via apical Na+ glucose, Na+ amino acid transporters and
NHEs. Absorption of Cl− by the proximal tubule occurs
via electro-neutral anion exchange coupled to the function
of NHEs (193). Absorption of Na+ and Cl− in the early
distal tubules occurs in part via NKCC2 while some paracellular Na+ transport driven by the serosal negative potential may also occur. The operation of NKCC may rely on
recycling of K+ across the apical membrane via K+ channels. Transport of Cl− across the apical membrane occurs via
K+ :Cl− cotransporters or conductive Cl− channels (193, 384,
1320). The reabsorption processes in the late distal tubules,
collecting tubules and collecting ducts of teleosts remain to be
characterized.
Role of the urinary bladder
The urinary bladder forms a final diluting segment comprised
of a high resistance, tight epithelium with exceedingly low
water permeability and capable of net absorption down to
about 2 mM NaCl (1162). In salmonids, Na+ and Cl− uptake
is electrically silent and largely independent of the counterion (225, 1161). NaCl absorption is sensitive to amiloride
but not affected by thiazide and bumetanide. Furthermore,
Na+ uptake in absence of Cl− is associated with acidification of the luminal solutions (225, 1164, 1166). These
observations all points to ion absorption via NHE and anion
exchangers.
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Osmoregulation and Excretion
Renal function and ambient salt concentrations
Given the quantitatively significant role of tubular NaCl
absorption for ion regulation by freshwater fish, the paucity
of data on the response of renal Na+ and Cl− transporters to
alterations of ambient NaCl concentrations is striking, especially considering the substantial effort devoted to branchial
transport processes. Renal transport processes in freshwater
fish are clearly subject to regulation depending on acid-base
status (598) and Na+ (668) balance, but nothing is known
about the nature of this regulation.
Marine Teleost Fish
Overview
Marine teleosts, being hypoosmotic with respect to the environment, experience a continuous diffusive water loss and ion
gain across the gill epithelium. Although urine flow is modest in marine teleosts when compared to freshwater species,
fluids lost both by urine and across the gill epithelium dictate a need for ingestion of seawater to maintain water balance
(651). The gastrointestinal tract is capable of water absorption
from ingested seawater, a process that ensures water balance
but leads to additional salt gain and thus a need for excretion
of mainly Na+ and Cl− , but also Mg2+ and SO4 2− . Since
fish kidneys are incapable of producing hyperosmotic urine,
monovalent ions are excreted mainly across the gill while
renal excretion of low urine volumes compensates for excess
Mg2+ and SO4 2− gain (1167).
Gills
Early studies identified “chloride cells” (now referred to as
MR cells) as the likely site of Cl− excretion by gills of
seawater-acclimated European eel (913, 914). More recent
studies, using the opercular epithelium of the killifish as a
model for marine fish gills, demonstrate that density of MR
cells and epithelial conductance is proportional to ambient
salinity and that the MR cells indeed are responsible for Cl−
secretion (370, 545). In seawater, pavement cells cover ∼90%
of the gill surface area, with MR cells occupying mainly the
trailing edge of the filament and the interlamellar regions
(726, 890). In addition to numerous mitochondria and an
extensive tubular network derived from basolateral invaginations, branchial MR cells of marine teleosts are characterized
by an apical crypt and are always associated with adjacent
accessory cells (890, 1461). MR cells and accessory cells
show extensive interdigitations, have shallow tight junctions
and are considered to be the site of the relatively high ionic
permeability of the marine teleost gill epithelium (509, 884,
1606).
Na+ and Cl− excretion The transport model for marine
teleost fish Na+ and Cl− excretion (Fig. 18) is better established than the models for Na+ and Cl− uptake by freshwater
fish. Excretion of Na+ and Cl− is driven by Na+ /K+ -ATPase,
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Comprehensive Physiology
AC
Water
TJ
Claudin 10e
Na+
MRC
–30 mV
~
Na+
K+
Cl–
eKir
CFTR
Na+,K+,2Cl–
NKCC1
Figure 18 Accepted transport processes in the branchial epithelium of marine teleost fish. MRC = MR cell, AC = accessory cell.
Transcellular Cl− secretion includes entry across the basolateral membrane by the secretory isoform of the Na+ -2Cl− -K+ isoform (NKCC),
which is driven by the electrochemical Na+ gradient established by
the Na+ /K+ -ATPase (∼), and exit across the apical membrane via
the cystic fibrosis transmembrane regulator (CFTR) Cl− channel. The
active secretion of Cl− establishes a transepithelial potential of −1740 mV (reference blood side), which drives paracellular Na+ secretion
through cation-selective “leaky junctions” between MRCs and ACs.
Cation selectivity of the junctions may be mediated by claudin 10e
and possibly other claudins. A basolateral inward rectifying K+ channel (eKir) allows for recycling across the basolateral membrane. The
depicted transport model also described Na+ and Cl− secretion by the
renal proximal tubules and the elasmobranch rectal gland. See the text
for further details.
which is highly abundant in the basolateral membrane (885,
2004, 2006), as ouabain inhibits not only enzyme activity but
also efflux of both ions (1417, 1690). The basolateral Na+ /K+ ATPase establishes a Na+ gradient allowing for entry of Cl−
and Na+ via the basolateral secretory Na+ -2Cl− -K+ cotransporter (NKCC1), for which the gene is expressed in proportion
to ambient salinity (367, 1098, 1168, 1417, 1836) leading to
corresponding protein levels (538, 882, 1605). Observations
of Ba2+ blocking the short-circuit current across killifish opercular epithelia (413), the cloning of an inward-rectifying K+
channel (Kir) from Japanese eel and observations of increased
expression of Kir during acclimation to seawater (1793) suggest that K+ is recycled across the basolateral membrane,
entering via NKCC1 and exiting via Kir, to allow for Cl−
and Na+ uptake. Once in the MR cells, Cl− is above thermodynamic equilibrium and exits across the apical membrane
via a CFTR-type channel. Teleost CFTR was first identified
in killifish (1165, 1696) and has since been identified in a
number of seawater teleosts. Gene expression (149, 1835)
and CFTR protein amount (1662, 1805, 1871) increase with
increasing salinity. In addition to transcriptional regulation,
CFTR function, at least in the euryhaline killifish, is subject
to translocation of protein from cytosolic compartments to the
456
apical membrane (1168) and is under regulation by phosphorylation of focal adhesion kinase (1169).
Cation selectivity of gills and opercular epithelia from
seawater-acclimated trout and killifish, respectively, provided
evidence that Na+ secretion across marine fish gill occurs
passively via a paracellular shunt pathway (414, 937, 1163).
The magnitude of the gill transepithelial potential (TEP) in
most seawater teleosts ranges from −17 to −40 mV (reference
in body fluids) (1167), which in many cases is insufficient to
drive Na+ secretion against the blood:water concentration
gradient. However, it is likely that the TEP measured in vivo
is less than that localized at the MR cells due to the large
surface area of pavement cells, which adds to the passive
shunt (1167). The gulf toadfish represents an exception by
having an outside positive gill TEP (506), suggesting unique
ion permeability of the gill and that Na+ secretion by this
species may not be passive.
To date, a single member of the claudin gene family,
claudin 10e of Atlantic salmon, shows increased branchial
expression following seawater transfer (1835), pointing to a
role of this tight junction protein in controlling the cation
permeability of the seawater acclimated fish gill.
MR cell diversity Like freshwater teleosts, marine
teleosts may possess distinct MR cell types; at least one
marine teleost has been reported to have one cell type expressing apical NHE2 and basolateral Na+ /K+ -ATPase, while
another expresses basolateral H+ -ATPase, and are likely
involved in acid and base secretion, respectively (263).
Gastrointestinal tract
Seawater ingestion and intestinal water absorption was
demonstrated in 1930 (1713), and it has since been established that drinking rates in marine and seawater-acclimated
euryhaline fish range from 1 to 5 ml/kg/h (254, 564-566,
568, 660, 663, 1061, 1663, 1800), 10-50-fold higher than in
freshwater fish (254, 1061, 1148, 1423). The majority (70%85%) of ingested seawater is absorbed by the intestine with
the remainder being excreted as rectal fluids (597, 747, 1663,
2009). Gastrointestinal processes, rather than branchial and
renal processes, involved in osmoregulation have recently
been demonstrated to be limiting for survival of teleost fish in
high-saline environments (596).
Control of drinking rates
Changes in drinking rates present the most immediate level of
control over fluid absorption by the gastrointestinal tract, and
control of the drinking rate is therefore central to osmoregulation by marine fish. Control of teleost fish drinking rates
have been reviewed recently (654, 1798) and includes a rapid
response to changes in ambient salinity, distension of the
gastric wall and intestine, as well as luminal salt concentrations (42, 769). Although it cannot be excluded that these
rapid responses are mediated by the renin-angiotensin system
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Comprehensive Physiology
(RAS, discussed below), it occurs within minutes and much
faster than the onset of significant hypotension or hypovolemia caused by dehydration (70). Ion replacement studies
demonstrated that Cl− is the trigger for externally stimulated
drinking and the negative feedback resulting from elevated
luminal Cl− concentrations in the intestinal lumen (42, 769).
RAS is clearly involved in regulation of drinking by teleosts
via angiotensin II (ANG II) acting on the so-called swallowing center in the medulla oblongata to initiate water ingestion (254, 563, 565, 1423, 1797, 1799, 1800). Hypovolemia
and hypotension as a result of vasodilation potently stimulate
RAS-induced drinking via elevated ANG II (565, 769). In
addition, bradykinin, the active component of the kallikreinkini system, and atrial natriuretic peptide both act on RAS
by altering ANG II levels to inhibit drinking, although it is
unknown whether these effects are mediated solely through
RAS (334, 335, 585, 1801, 1872).
Esophageal desalinization
Around 50% of NaCl ingested with seawater is absorbed by
the relatively short esophagus in a 1Na+ :1Cl− ratio (651).
This high NaCl absorption rate, combined with low water
permeability (770, 1403), explains the marked reduction in
osmotic pressure of ingested seawater (∼1000 mOsm) as it
travels to the gastric lumen (∼500 mOsm) (930, 1403, 1713,
2009). Passive and active absorption of Na+ and Cl− occur
in the esophagus, but it appears that the esophagus is relatively impermeable to other ions and water. For the few
species examined, Na+ uptake occurs via Na+ /H+ exchange
or Na+ channels (amiloride sensitive) rather than NCC or
NKCC2 cotransporters (770, 1403), while the mechanisms of
Cl− uptake remain unknown.
Absorption by the intestine
Intestinal water absorption in marine teleosts is tightly linked
to absorption of Na+ and Cl− (1123, 1701, 1888) with >95%
of the ingested NaCl being absorbed (597). The absorbate is
hyperosmotic (651) and the resulting net salt gain is compensated by branchial excretion (discussed above). Ion absorption
in the anterior segment of the intestine results in isoosmotic
fluids in the intestinal lumen, allowing for solute coupled
water absorption (651, 654, 662, 665). Entry of Na+ across
the apical membrane occurs via two cotransport pathways,
NKCC2 and NCC (367, 530, 560, 687, 688, 1266) (Fig. 19).
Considering the Na+ and H+ gradients across the apical membrane, both Na+ -channels and NHEs may contribute to Na+
uptake from the lumen, but at present no evidence has been
reported for involvement of Na+ channels. However, elevated
NHE3 mRNA expression in rainbow trout intestinal mucosa
during acclimation to seawater (658) suggests a role for this
NHE in intestinal Na+ absorption, although luminal amiloride
in the pyloric caecae and anterior regions does not appear to
reduce H+ secretion (657). A role for NHEs in intestinal
Na+ absorption was also suggested by in situ gut perfusion
experiments on seawater-acclimated rainbow trout showing
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Osmoregulation and Excretion
reduced acid-secretion in the presence of luminal amiloride
(2009).
Transport of Na+ across the basolateral membrane occurs
via Na+ /K+ -ATPase that is highly abundant in the basal
and lateral membranes of the columnar enterocytes (653).
The intestinal epithelium shows the highest Na+ /K+ -ATPase
activity of the three osmoregulatory organs (655, 786) and
mRNA expression of the α-subunit of the pump, as well as
enzymatic activity is most often higher in seawater-acclimated
compared to freshwater-acclimated euryhaline species—a
trend that also applies to NKCC2 mRNA levels (328, 567,
838, 909, 1141, 1142, 1644, 1836). In addition to fueling
apical entry of Na+ , the Na+ /K+ -ATPase also energizes Na+
transport processes across the basolateral membrane. Basolateral H+ extrusion occurs via a Na+ -dependent mechanism,
presumably NHE1 (595, 656), and basolateral Na+ :HCO3 −
cotransport is important for high rates of intestinal HCO3 −
secretion (see below; [658, 1813]).
The intestine displays net K+ absorption. Considering K+
concentrations in seawater and the intestinal fluids are 10 and
∼5 mM, respectively (661), and the substantial water absorption, it is clear that the majority of the ingested K+ is absorbed.
Undoubtedly, NKCC2 is involved in K+ absorption, but considering the concentrations of K+ , Na+ and Cl− in seawater, it becomes clear that K+ might limit NKCC2 function.
Observations of a Ba3+ -sensitive K+ channel (1266) suggests
that K+ may recycle across the apical membrane to facilitate
transport by NKCC2 as is seen for NKCC1 in the basolateral
membrane of the marine teleost gill epithelium (see above).
A basolateral K+ :Cl− cotransporter facilitates K+ movement
across the basolateral membrane (1711).
The net absorption of Cl− , like Na+ , occurs against an
electrochemical gradient (Table 6 and Fig. 19). However, in
contrast to Na+ , which enters across the apical membrane
down an electrochemical gradient, Cl− uptake across the apical membrane occurs against an electrochemical gradient.
Cl− movement from the cytosol across the basolateral membrane is following the electrochemical gradient via K+ :Cl−
cotransport or Cl− -channels, including a CFTR paralogue
(1097, 1168, 1711). At least two distinct pathways contribute
to apical Cl− entry in the marine teleost intestinal epithelium.
One pathway relies on the inward-directed electrochemical
Na+ gradient via cotransporters (NCC and NKCC2) discussed above, while the other pathway is via Cl− /HCO3 −
exchange. The evidence for the presence of these two parallel
pathways, which are both quantitatively important, has been
discussed in detail in previous reviews (651-654, 661, 662,
665). High HCO3 − concentrations in intestinal fluids were
first inferred in 1930 (1713) and have since been documented
for a high number of marine or seawater-acclimated euryhaline fish (651, 661, 1814, 2008), and are the result of highactivity apical Cl− /HCO3 − exchange proteins in the apical
membrane. Convincing correlations between Cl− uptake and
apparent HCO3 − secretion across the intestinal epithelium of
a seawater-acclimated teleost was first reported by Shehadeh
and Gordon (1663) and was attributed to anion exchange,
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Osmoregulation and Excretion
Comprehensive Physiology
TJ
CaCO3
CO32–
Na+
H+
Lumen
HCO3–
~
NHE1
H+
CO2
HCO3–
K+
Na+
CAc
CO2+H2O
CAIV
nHCO3–
HCO3–
HCO3–
NBC1
A6
CO2
Cl–
H+
Na+
~
+
H
NHE2
Na+
H+
H+
NHE3
Na+
CAc
HCO3–
+
NKCC2
NCC
sAC
Na+, K+, 2Cl–
Na+, Cl–
K+
+20 mV
~
0 mV
~
TJ
H+
Claudins
3, 15, 25b
Na+
H2O
lis
1
Cl–
H2O
mosmol/kg 650, pH 1-2
P
AQ
H2O
AQP1
Figure 19 Accepted (solid black) and putative (gray) transport processes in the intestinal epithelium of marine
teleost fish (redrawn from [652, 654, 662]). Water transport, transcellular (potentially via AQP1) and/or paracellular (dotted lines) is driven by active NaCl absorption providing a hyperosmotic coupling compartment in the lateral
interspace (lis). Entry of Na+ across the apical membrane via cotransporters (NKCC2 and NCC) and extrusion
across the basolateral membrane via Na+/K+-ATPase accounts for transepithelial Na+ movement. In addition,
recent evidence that NHE2 and NHE3 are expressed in the intestinal epithelium suggested that Na+ uptake across
the apical membrane may occur also via these transporters. Entry of Cl− across the apical membrane occurs via
both cotransporters and Cl− /HCO3 − exchange conducted by the SLC26a6 anion exchanger while Cl− exits the
cell via basolateral anion channels. Cellular HCO3 − for apical anion exchange is provided in part by HCO3 − entry
across the basolateral membrane via NBC1 and in part by hydration of endogenous metabolic CO2 . Cytosolic carbonic anhydrase (CAc) found mainly in the apical region of the enterocytes facilitates CO2 hydration. (Continued)
458
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Comprehensive Physiology
which accounts for continued Cl− uptake in absence of
luminal Na+ (655, 661). Recent reports identified at least
one anion exchange protein, SLC26a6, to be important for
Cl− /HCO3 − exchange across the apical membrane (662, 982).
Interestingly, this anion exchanger operates in a Cl− /nHCO3 −
exchange mode, is therefore electrogenic and is fueled in
part by the apical membrane potential to allow for transport
of Cl− and HCO3 − against seemingly unfavorable chemical
gradients (662).
The salt absorption described above facilitates water
absorption despite the lack of net osmotic gradients. The
absorbate is significantly hypertonic (651), and it appears that
the lateral interspace (lis) between enterocytes acts as a coupling compartment for salt and water absorption (Fig 19). Salt
transport across the lateral membranes renders the fluids in lis
hyperosmotic, which drives water movement. Water absorption in the direction from the lumen to the blood is likely
ensured by barrier properties of the tight junctions, resulting
in dispersal of salts and thereby water in the direction from
lis to the blood side of the epithelium. See recent reports tor
detailed accounts of solute-coupled water movement across
vertebrate epithelia (997-1000, 1301). It is unknown whether
water enters the lis from the intestinal lumen across the tight
junctions or by a transcellular pathway, although a recent
study points to water movement mainly by a transcellular
route (2026). Of the three aquaporins (AQPe, AQP1 and
AQP3; [49, 368, 1051, 1176, 1177, 1500]) observed to be
expressed in the marine teleost intestine, AQP1 may play
a role in water absorption. Observations of elevated mRNA
expression following seawater transfer, elevated AQP1 protein abundance in intestinal tissue following seawater acclimation and finally apical as well as basolateral localization of
the protein suggests a role in transepithelial water movement
(49, 1176, 1177, 1500). It has been demonstrated that AQP1
confers water permeability in expression systems, but despite
the above observations, unequivocal evidence for a role for
AQP1 in intestinal water absorption is lacking.
Exclusion and secretion by the intestine
Luminal concentrations of Mg2+ and SO4 2− are typically
120-150 mM and 100-120 mM, respectively, due to selective
Osmoregulation and Excretion
absorption of Na+ , Cl− and water (Table 6 and [596, 597,
651, 661, 1167]). As plasma concentrations of these divalent
ions are < 1 mM, the gradient from the intestinal lumen to
the extracellular fluids is greater than it is for any other ions.
With this in mind, it is remarkable that ∼90% of the ingested
MgSO4 passes through the intestinal tract unabsorbed (597,
747, 2010). Nevertheless, some Mg2+ and SO4 2− are absorbed
owing to the considerable gradient across the intestinal (and
branchial) epithelium. The resulting excess of these divalent
ions is cleared by renal excretion, as discussed below. Limited intestinal SO4 2− absorption is not strictly a matter of
barrier functions as SO4 2− secretion via anion exchange for
Cl− uptake acts to reduce overall net SO4 2− uptake (1415).
The nature of the anion exchanger responsible for intestinal
SO4 2− secretion remains unknown, but recent studies demonstrated that SLC26a6 is responsible for SO4 2− /Cl− exchange
by renal tubules of marine teleosts (889). Since urine and
intestinal fluids of marine teleosts are similar with respect
to Cl− and SO4 2− concentrations (Table 6), it is possible that
SLC26a6 acts to secrete SO4 2− as well as HCO3 − in exchange
for Cl− uptake. The situation is even less clear for Mg2+ as it
is unknown whether the substantial gradients are maintained
strictly by barrier functions or if intestinal Mg2+ secretion
contributes to the very low net uptake. Recent findings show
changes in salinity alter mRNA expression of multiple isoforms for claudin 3, 15 and 25b in the intestinal epithelium of
euryhaline fish (308, 1838), which suggests that these claudins
may be involved in controlling and regulating the paracellular pathway and thus potentially the barrier functions of the
epithelium against movement of divalent ions. Of particular
interest is that certain isoforms are expressed more robustly
in the distal segments (308) where luminal-serosal gradients
are the steepest (1167).
In contrast to Mg2+ and SO4 2− , Ca2+ concentrations are
typically low (<5 mM) in intestinal fluids of marine fish.
However, these low concentrations are not due solely to
intestinal Ca2+ uptake but are the product of CaCO3 precipitate formation facilitated by the high luminal HCO3 − concentrations and alkaline conditions (2010, 2012). Precipitate
formation is a consistent phenomenon among marine fish and
has recently been demonstrated to contribute significantly to
oceanic CaCO3 production (2011). Although the fraction of
←
Figure 19
(Continued) Protons arising from CO2 hydration are extruded mainly across the basolateral membrane by a Na+-dependent
pathway and possibly by vacuolar H+ pumps. Some H+ extrusion occurs across the apical membrane via H+ pumps and possibly via NHE2
and/or NHE3 and masks some of the apical HCO3 − secretion by HCO3 − dehydration in the intestinal lumen yielding molecular CO2 . This
molecular CO2 may diffuse back into the enterocytes for rehydration and continued apical anion exchange. Furthermore, molecular CO2 from
this reaction is rehydrated in the enterocytes and resulting HCO3 − is sensed by soluble adenylyl cyclase (sAC), which appears to stimulate ion
absorption via NKCC2 (+)(1859). Conversion of HCO3 − to CO2 in the intestinal lumen is facilitated by membrane-bound carbonic anhydrase,
CAIV and possibly other isoforms, a process that consumes H+ and thereby contributes to luminal alkalinization and CO2 − formation. Titration
of luminal HCO3 − and formation of CO2 − facilitates formation of CaCO3 precipitates to reduce luminal osmotic pressure and thus aid water
absorption. SLC26a6, the electrogenic anion exchanger, exports nHCO3 − in exchange for 1Cl− , and its activity is therefore stimulated by the
hyperpolarizing effect of the H+ pump. The apical electrogenic nHCO3 − /Cl− exchanger (SLC26a6) and electrogenic H+ pump constitutes
a transport metabolon, perhaps accounting for the apparently active secretion of HCO3 − and the uphill movement of Cl− across the apical
membrane. The indicated values for osmotic pressure and pH in the absorbed fluids are based on measured net movements of H2 O and
electrolytes including H+s, but the degree of hypertonicity and acidity in lis likely are much less than indicated due to rapid equilibration with
subepithelial fluid compartments. “TJ” = tight junction. Selective permeability of the epithelium is likely related to multiple isoforms of claudin 3,
clauding 15 and claudin 25b, although other claudins may also be involved. See text for further details.
Volume 4, April 2014
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Osmoregulation and Excretion
ingested Ca2+ taken up by the intestinal epithelium differs
among reports and possibly species, the formation of precipitates in the intestinal lumen acts to reduce osmotic pressure
by up to 70 mOsm as Ca2+ and HCO3 − are removed from
solution. A reduction of osmotic pressure of this magnitude
is significant for water absorption and thus osmoregulation
(1983, 2012). Facilitation of CaCO3 formation in the intestinal
lumen is possibly enhanced by the activity of an extracellular,
apical-membrane-bound carbonic anhydrase isoform (possibly CA-IV) (657, 658).
Secretion of HCO3 − by the teleost intestine correlates
with ambient salinity and is temperature dependent (656) but
is generally in the order of 0.5 μmol/cm2 /h under resting,
physiological conditions (43, 436, 570, 656, 657, 661, 662,
667, 1813, 1815, 2012). This rate is comparable to resting
secretion rates by the mammalian duodenum at 37◦ C (301,
785, 1877, 1878); the high rates of HCO3 − secretion lead to
luminal concentrations of up to 100 mM (651, 662, 2012).
Transepithelial HCO3 − transport as well as hydration of
endogenous CO2 within the intestinal epithelium both contribute to the high rates of secretion, although to varying extent
in different species (43, 436, 656, 657, 667, 2010). Considering transepithelial transport, entry of HCO3 − across the basolateral membrane takes place via an electrogenic Na+ :HCO3 −
cotransporter (NBC1), for which expression is dependent on
ambient salinity (982, 1813). Intestinal cytosolic carbonic
anhydrase (CAc) catalyzes the hydration of endogenous CO2
providing HCO3 − for apical anion exchange; CAc mRNA
expression in intestinal tissues also shows elevated mRNA
expression following transfer to elevated salinity (658, 1610).
CAc localizes to the apical region of the intestinal epithelium (658), and application of permeant CA inhibitors inhibits
luminal HCO3 − secretion (436, 657, 667, 2009), demonstrating an important role for this enzyme in apical anion exchange.
Intestinal HCO3 − secretion in the absence of serosal HCO3 −
and CO2 remains high in all species tested and leads to the
prediction of high CO2 production by the intestinal epithelium, which was confirmed by findings of intestinal tissue
mass-specific metabolic rates being threefold higher than corresponding whole animal rates (1815). Furthermore, conversion of CO2 to HCO3 − is highly efficient as the majority of
metabolic CO2 is converted to HCO3 − and secreted across
the apical membrane (1815).
The high CO2 hydration rates in the intestinal epithelium
generate protons that must be secreted from the cells to maintain intracellular pH and continued CO2 hydration. Secretion
of protons occurs mainly across the basolateral membrane
as the tissue exhibits high net base secretion rates and basolateral H+ -secretion has been measured directly. Inhibition of
basolateral H+ extrusion by removal of serosal Na+ and application of ouabain (656) implicates basolateral NHE1 that is
present in seawater-acclimated rainbow trout (595) and illustrates that Na+ /K+ -ATPase indirectly drives active transport
of HCO3 − (and Cl− ) across the apical membrane (656).
In addition to NHE1, the vacuolar H+ -ATPase is present
in the basolateral as well as the apical membrane (658)
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Comprehensive Physiology
and likely contributes to H+ -secretion across the basolateral membrane, although this remains to be demonstrated.
In contrast, acid secretion across the apical membrane by the
vacuolar H+ pump has been demonstrated for at least two
species (657, 662) and appears to be elevated in fish exposed
to elevated salinity and most pronounced in the distal segments of the intestine (675). Apical H+ secretion may seem
counterintuitive in a net base secreting epithelium but likely
serves osmoregulatory purposes. One consequence of apical
H+ secretion is greater potential for cytosolic HCO3 − accumulation, in particular in the apical region rich in CAs and
thus more favorable HCO3 − gradients for continued anion
exchange. In addition, apical H+ pump activity will act to
further polarize the apical membrane, which will also act to
enhance anion exchange by the electrogenic SLC26a6. Thus,
the H+ pump contributes to intestinal Cl− uptake and thereby
solute-coupled water absorption. Finally, secretion of acid
into the intestinal lumen will act to titrate and thus reduce
the concentration of HCO3 − in luminal fluids and thereby the
osmotic pressure. Such reduced luminal osmotic pressure will
further assist water absorption. It is important to appreciate
that apical H+ secretion does not prevent or reduce CaCO3
formation in the lumen because resulting HCO3 − concentrations are far in excess of luminal Ca2+ concentrations (see
[652, 654, 662] for detailed discussions).
Kidney
Regardless of whether glomeruli are present, marine teleosts
produce low urine flow rates of 0.03-0.9 ml/kg/h (115, 542,
749, 1203, 1204, 1206, 1208, 1713). Fish are incapable of
producing concentrated urine, and while freshwater fish urine
is dilute, urine of marine teleosts is isoosmotic to the extracellular fluids, with Mg2+ , SO4 2− and Cl− being the major
electrolytes (115, 193, 747, 1204, 1206, 1663) (Table 6).
Tubule anatomy and function
Nephrons of the aglomerular kidney, which have been
reported for ∼ 30 teleost species, lack a renal corpuscle
(115, 123). However, the majority of marine teleosts possess
a glomerular nephron with a renal corpuscle containing the
glomerulus. Common for marine teleost fish with glomerular
and aglomerular kidneys is that almost all lack a distal nephron
(384), which is responsible for Na+ and Cl− reabsorption in
freshwater fish and other vertebrates (749). The tubules consist of two or three proximal segments that connect directly to
the collecting duct via the connecting tubule (115, 123). The
fact that loss of glomeruli has occurred several times during
evolution and that urine composition from marine glomerular
and aglomerular teleosts is near identical, illustrates that the
renal contribution to marine teleost osmoregulation, regardless of the presence or absence of glomeruli is dominated
by tubular secretion (115, 116, 118, 121, 123). Nevertheless,
species with glomerular kidneys display filtration rates of
∼0.5 ml/kg/h (see above).
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Comprehensive Physiology
Renal tubule secretion Na+ and Cl− are the dominants electrolytes in fluids secreted by isolated tubules from
glomerular as well as aglomerular species. However, Mg2+
and SO4 2− concentrations in tubular secretions are elevated
far above plasma concentrations and thus may also contribute
to fluid secretion (120, 309, 310). Tubular secretion of Na+
and Cl− is driven by Na+ /K+ -ATPase and presumably occurs
much like it does across the gill (Fig. 18), with basolateral
entry via NKCC1, paracellular movement of Na+ and apical Cl− secretion via cAMP-stimulated anion channels yet
to be identified (120, 311). Although Mg2+ concentrations
of 20 mM in tubular secretions (116, 121, 313, 1522) are
lower than the ∼140 mM observed in bladder urine (80, 596,
749, 1224), tubular Mg2+ secretion must be the product of
active transport. Entry of Mg2+ across the basolateral membrane is thought to occur via Mg2+ channels with active transport across the apical membrane via Mg2+ :Na+ exchange or
Mg2+ :H+ exchange driven by Na+ /K+ -ATPase and the H+ pump, respectively (115, 1295, 1527). Like Mg2+ , SO4 2− is
also subject to tubular secretion, although bladder urine concentrations (596, 1203) by far exceed those observed in tubular secretions (10 mM) (116, 313). Regardless of glomeruli,
active tubular SO4 2− secretion contributes the most to renal
excretion (124, 426, 1524) and is facilitated by basolateral
and apical anion exchange as well as cytosolic carbonic anhydrase (1414, 1525, 1526). Basolateral SO4 2− :OH− exchange
mediates SO4 2− import while SO4 2− /HCO3 − or SO4 2− /Cl−
exchange takes place across the apical membrane (1416).
Most recently, the nature of the transporter responsible for
apical secretion was identified to be an anion exchanger of the
SLC26 family, an electrogenic SLC26a6 isoform (889), identical to the transporter responsible for Cl− /HCO3 − exchange
in the apical membrane of the marine teleost intestinal epithelium (see above).
Renal tubular and urinary bladder absorption
The discrepancy between Mg2+ and SO4 2− concentrations in
tubular secretions and urinary bladder urine, discussed above,
is the product of fluid absorption driven by Na+ and Cl−
reabsorption in the distal proximal tubules and the urinary
bladder, which results in low rates of urine flow and highly
concentrated MgSO4 (1167). Average glomerular fluid secretion rates are comparable to urine flow rates in marine teleosts,
but fluid secretion rates may exceed glomerular filtration by
as much as three- to four times (115). Thus, tubular fluid
absorption rates are significant. The Na+ /K+ -ATPase creates
favorable gradients for tubular Na+ -glucose and Na+ -amino
acid cotransport, as well as Na+ /H+ exchange coupled with
apical Cl− /HCO3 − exchange (193, 384). The urinary bladder
contributes to Na+ , Cl− and water absorption and is responsible for absorption of as much as 60% of the urine in vivo
(806). In contrast to the proximal tubule, Na+ and Cl− absorption in the urinary bladder is coupled and occurs via Na+ :Cl−
cotransporters resulting in absorption of isoosmotic fluid (577,
1096, 1521, 1523). Urotensin I and II secreted from the caudal
Volume 4, April 2014
Osmoregulation and Excretion
neurosecretory urophysis may control transport by the urinary
bladder epithelium (112).
Amphibia
The Amphibia were the first vertebrates that made the transition to land still depending, however, on the aquatic environment for reproduction. All modern amphibians belong to
the subclass Lissamphibia that includes three orders: Anura
(frogs and toads), Caudata/Urodela (newts and salamanders)
and Gymnophiona (apodans or caecilians). Anuran species
amount to 88% of a total of 6,771 recognized species within
the subclass (561). The section on amphibian osmoregulation provides new perspectives for the combined role in ion
transport by principal and MR cells of the epidermis and the
subepidermal mucous glands as adaptations for aquatic and
terrestrial life. With the kidneys responding fast to change
in the availability of environmental water, the urinary bladder being a water storage organ, and anatomical, physiological and behavioral specializations for cutaneous drinking,
amphibians are well adapted for prolonged stay on dry land.
The putative selective pressure driving this evolution is an
expanded niche exploration, and except for polar and highaltitude regions, modern amphibians are found in all climatic
zones, and species from all three orders occupy a range of
habitats from purely aquatic to purely terrestrial (456, 1756).
While the natural history is well known for a large number
of amphibian species, the physiology has been studied for a
relatively small number belonging predominantly to the anurans. Aspects of amphibian osmoregulation are reviewed in
(759, 764, 860, 1792, 1881).
Extracellular and Intracellular Ion
Concentrations
In all three orders of amphibians the extracellular osmolality
and the concentrations of Na+ and Cl− are lower than those
of other vertebrates (see Table 7). The much larger concentrations, above 500 mosmol/kg H2 O, stem from burrowing
desert species and species adapted to survive or live in coastal
waters and estuaries (630, 894, 896, 1193). The extracellular K+ concentration spans a remarkably wide range from
less than 3 mM in B. marinus to 9 mM in R. cancrivora
and Ambystoma with the lower values being characteristic
of most studies. The high extracellular K+ may reflect loss
of K+ from skeletal muscles because exercise (and anoxia)
elevates plasma K+ (1933). Among anurans, the concentrations of Na+ and Cl− vary between species and laboratory
conditions. Amphibian plasma pH is between 7.8 and 8.1.
The HCO3 − /pCO2 system is the major extracellular buffer
and with pulmonary gas exchange and an arterial pCO2 of
∼1.6 kPa, the associated HCO3 − is about 22 mM (161). In
freshwater with cutaneous gas exchange or gill respiration
the arterial pCO2 is in the range of 0.4-0.7 kPa, resulting
in plasma HCO3 − of 7-10 mM at physiological extracellular
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Osmoregulation and Excretion
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Table 7
Osmolality (π , mosmol/kg H2 O) and Concentration of Diffusible Ions and Urea (mmol/l) of Blood Plasma or Lymph as Indicated of
Some Amphibians (mean ± sem or Range, Unless Otherwise Indicated)
Blood plasma or lymph
Species
π
Na+
K+
HCO3 −
Cl−
Urea
Remarks
11±2
In tap water
Anura
Rana ridibunda
(892)
247±12
115±5
6±1
83±6
Rana pipiens
(1261)
193±8
112±3
Rana cancrivora
(630)
290±10
125±17
9±1
98±10
Rana
catesbeiana
(1769)
214.6±7.5
100.2±5.1
2.5±0.6
58.5±6.5
Bufo boreas
(1261)
235±15
109±3
Bufo viridis
(1679)
275±5
120±6
4.0±0.4
87±2
Bufo bufo
(1308)
119±2
3.0±0.25
79±2
In tap water, lymph
samples, mean±sd
Bufo bufo (847)
105.5±1.0
85.4±2.6
Terrestrial habitat
free access to tap
water and
mealworms
68±3
Semiaquatic, kept in
about 0.2 mM NaCl
40±1
In fresh water
mean±sd 20◦ C
27.1
77±5
Terrestrial, collected
in the field
17±2
In tap water
Lymph samples
234±10
107.9±6.8
2.4±0.4
76.1±4.9
Bufo marinus
(960)
241.2±3.4
99.8±2.6
4.6±0.1
72.6±3.8
Bufo marinus
(586)
209.4±3.9
126.7±2.4
3.64±0.20
—
Distilled water
adapted
Bufo marinus
(586)
300.2±3.8
185.5±4.7
3.59±0.28
—
Saline adapted
0.9% NaCl
Hyla regilla
(1261)
218±10
110±3
78±5
Terrestrial, collected
in the field
Ascaphus truei
(1261)
172±5
106±3
81±3
High altitude
streams, collected in
the field
Scaphiopus
couchi (1193)
225-390
253-346
141-210
120-174
3.6-14.4
3.6-5.7
84-152
92-125
37-173
18-51
Collected from
temporary pools
590
630
204
228
5.4
5.4
159
165
228
286
2 toads emerging
from ground
borrows
290±10
125±17
9±1
98±10
40±1
In fresh water
830±50
252±12
14±0.5
227±9
Rana cancrivora
(630)
Bufo viridis
(894)
(896)
462
24.1
—
mean±sd 20◦ C
Bufo marinus
(1264, 1769)
19.6±3.2
350±1
In 80% saltwater
296±17
22.2±12.3
In tapwater
597±11
158±50
In 580 mosmol/kg
NaCl (mean±sd)
392±15
141±6
110±10
32±13
Access to tap water
752±27
162±8
162±10
272±16
Burrowing
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Comprehensive Physiology
Table 7
Osmoregulation and Excretion
(Continued)
Blood plasma or lymph
Species
π
Na+
K+
Cl−
HCO3 −
Urea
Remarks
Caudata
Ambystoma gracile larvae
(1764)
94.4±2.4
9.5±0.6
75.4±1.7
Ambystoma tigrinum
(1670)
106
9
98
Ambystoma tigrinum
larvae (730)
107.9±1.3
5.2±0.3
Necturus maculosus (730)
93
2.5
Batrachoseps attenuates
(854)
191±2
339±8.9
In tap water at 15 ◦ C for
≥ 1 week unfed for 2 weeks
In tap water at 5 ◦ C, unfed for
≤2 months
82
110±7.0
48±8.2
Tap water at 15 ◦ C unfed
< 1 week
Gymnophiona
Typlonectes
compressicauda
(1765)
196.3±3.2
111.4±5.3
5.4±0.5
69.7±4.8
In tap water at 25 ◦ C, fed
Tubifex worms
Ichthyophis kohtaoensis
(1765)
220±5
101.3±9.0
3.2±0.3
87.5±1.9
Housed in damp potting soil at
room temperature, access to
live crickets
Ichthyophis kohtaoensis
(1765)
210.6±4.8
111.9±3.6
2.7±0.3
79.5±2.7
In tap water
pH of about 7.8 (230). The distribution of diffusible ions
between extracellular and intracellular fluids conforms to the
usual picture: cellular Na+ is significantly below and cellular
K+ above thermodynamic equilibrium (Table 8). In principal cells of the epidermis and acinar cells of subepidermal
exocrine glands, Cl− is above thermodynamic equilibrium,
whereas in striated muscle cells and in MR cells under terrestrial conditions Cl− is passively distributed. In freshwater
the electrochemical potential of Cl− would be above that of
the environment because of active uptake across the apical
membrane of MR cells.
Body Water Volumes
Amphibians contain relatively more water than do other vertebrates (Table 9). The total body water constitutes 70%-80%
of the body mass of which the intracellular fluid amounts to
70%-80% of the total water. The extracellular fluid is divided
between interstitial fluid, blood plasma and the lymphatic
system. It is an intriguing aspect of anuran osmoregulation
that they are tolerant to significant perturbations of body
water volume. Among the species studied, the extracellular
water volume ranges from 24% to 48% (Table 9) associated
Volume 4, April 2014
with a highly variable lymph volume, with little variation
in plasma volume (hematocrit), and thus unimpaired circulation (753, 757, 760, 781, 868, 1193). This owes to a fast
exchange of fluid between blood plasma, interstitial, and
lymph volumes (79, 332, 333, 755, 758, 1220) governed by a
high whole-body systemic transcapillary filtration coefficient
(692). The tolerance to desiccation of anurans is to some
degree correlated with their terrestriality (755, 756, 1829),
with the limit set by the volume loss impairing the circulatory system caused by an increased hematocrit (1670). The
limit may be surprisingly high as indicated in a study of
the aquatic Xenopus laevis; at an upper-limited/non-tolerated
water loss of 33.8±1.0% of the body mass, the extracellular osmolality was 523±14 mosmol/kg with a hematocrit of
65.5±2.4% (754).
Epidermal Epithelium of the Skin
The skin of amphibians is a major site for ion and water
exchange. Extensive studies have been performed on adult
stages, mostly of anurans as discussed below, but larval stages
also have been investigated (432, 911, 940, 941, 943). In larval stages, gills constitute an additional site for ion and water
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Table 8
Intracellular Concentrations of Small Diffusible Ions and Membrane Potentials of Striated Muscle Fibre and Anuran Epithelial Cells with
Osmoregulatory Function
Na+
K+
Tissue
Cl−
HCO3 −
mmol/l
Vm
mV
Striated muscle fibre
10.41
1241
1.51
12.41
−952
Acinus cells of subepidermal
mucous gland
11.53
1553
553
—
−69.5±0.74
Principal cell compartment of
epidermis, bilateral Ringer’s
solution
9.95
1595
495 , 506
—
−108±27
Mitochondria-rich cells of
epidermis, bilateral Ringer’s
solution
30.08
1508
47.98 38.4±3.99 1410 ,
2511 , 19.8±1.712
—
−33.2±2.59
Principal (granular) cells,
toad urinary bladder
19.513
16313
57.513
—
−9414 −57.2±2.315
Mitochondria-rich cells, toad
urinary bladder
11.2±2.516
114.4±3.716
—
—
−
1 (336). 2 (1231). 3 X-ray
microanalysis (774). 4 Basolateral membrane potential of resting gland cells (1733). 5 X-ray microanalysis (1545), here
and in the following corrected for gram dry mass per 100 g wet mass according to the information given in the respective papers. 6 (1126).
7 Basolateral membrane potential (1274). 8 X-ray microanalysis (1542). 9 GHK fit of single-channel i /V relationships in cell attached patch on cells
Cl
in free suspension, i.e., short circuited cells (1732).10 X-ray microanalysis (1546). 11 X-ray microanalysis (1543). 12 From estimated intracellular
−
13
Cl space of MR cells exposed to bilateral NaCl Ringer’s solution (1007). X-ray microanalysis (1544). 14 Basolateral membrane impalement
of short-circuited cells (1284).15 Basolateral membrane impalement of short-circuited cells (439).16 X-ray microanalysis in mmol/kg wet mass,
i.e., here the concentration is not corrected for dry mass of analyzed sections (1544).
exchange (64, 98, 1039-1043), but the quantitative role in
maintaining larval ion balance is not yet fully established.
Amphibians do not drink orally; the colon epithelium, however, expresses luminal Na+ channels that are regulated like
the apical Na+ channel of the skin (253, 326, 399, 965-967,
1038). Under terrestrial laboratory conditions, whole-body
ion balance of Na+ and Cl− in B. boreas and R. pipiens
depends on ingested food (759). Generally, for aquatic environments quantitative studies of the relative significance of
dietary and cutaneous ion intakes are lacking.
Pioneered by Hans Ussing, in several years following
World War II, frog skin served as one of the preferred experimental preparations in the study of active ion transport and
functional organization of transporting epithelia (991, 992).
The first model of a NaCl-absorbing epithelium was developed for frog skin (956), which influenced studies of ion
transport by epithelia with osmoregulatory functions generally (508, 1388). The micrograph of Fig. 20 shows that anuran skin comprises a heterocellular, multilayered absorbing
epithelium with principal cells and MR cells (219, 220, 484,
520, 1742, 1980), and subepidermal glands (495, 1230, 1322,
1323). Numerous studies on amphibian skin have extended
the original model, which today includes observations on
a large number of species considering additional ion exchange pathways, water channels, regulatory mechanisms and
functions of the subepidermal glands. Figure 21 shows
464
updated models of the functional organization of anuran epidermis. Because of the transitional position of adult amphibians, the models of Fig. 21 indicate adaptations both for
the life in freshwater and on land as reflected by the apical aquaporin water channels of principal cells and pathways
for both active and passive Cl− uptake of MR cells. Further
to this, regulatory mechanisms at cell level have evolved for
meeting a lifestyle that comprises both aquatic and terrestrial adaptations. Each of the three transporting units shown
in Fig. 21—(i) principal cells, (ii) MR cells and (iii) subepidermal glands—is configured for specific functions related to
amphibian osmotic and ionic regulation. They are dealt with
in separate paragraphs below, which also discuss the functional interplay between the two epidermal cell types as well
as the coupling of epidermal and subepidermal functions in
osmoregulation.
Principal cell compartment
The multilayered principal cell compartment that amounts
to 90%-95% of the total epidermis is specialized for active
uptake of Na+ (1894). The cells of all layers constitute a functional syncytium enclosed by the apical plasma membrane of
the outermost living cell layer and by the plasma membranes
lining the labyrinth of intercellular spaces (519, 520, 1273,
1545, 1656, 1718, 1893). Gap junctions are included in this
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Comprehensive Physiology
Osmoregulation and Excretion
Table 9
Amphibian Body Fluid Volumes Expressed as Percent of Total Body Mass (Hematocrit: % Blood Cells). Where Indicated, Total Body
Water (TBW) is Calculated Relative to the Mass with the Urinary Bladder Emptied, Which Defines a “Standard Body Mass” (1671)
Species
TBW
Extracellular
vol. Marker
Plasma vol.
Marker
Hematocrit
% cells
Rana pipiens
78.91
23.6-29.82
5.6-9.32
Thiocyanate
T-1824
Rana catesbeiana
79.0±0.511
29.8-47.92
7.2-8.82
30±5.33
Thiocyanate
3.7±0.311
22-3411
21.7±1.011
T-1824
Remarks
Ref. (1827) unfed in tab
water for ≥ 2 weeks
Sucrose
Hyla cinerea
80.11
Scaphiopus hammondii
80.01
Scaphiopus couchi
69.8-81.64
Aneides flavipunctatus
71-785
Bufo bufo
81.3±1.06∗
Bufo marinus
74.1±0.711
24.7±1.511
Sucrose
4.7±0.511
T-1824
Bufo viridis
72.7±0.98
358
14 C-inulin
6.38
Evans blue
Necturus maculosus
81.1±0.511
24.1±0.711
Sucrose
4.7±0.211
28-3811
Ref. (1827) unfed in tab
water for ≥ 2 weeks
Cryptobranchus
alleganiensis
79.1±0.511
22.0±1.011
Sucrose
3.5±0.1611
T-1824
36-4511
Ref. (1827) unfed in tab
water for ≥ 2 weeks
Amphiuma means
78.8±0.311
21.8±0.811
Sucrose
3.4±0.211
T-1824
18-3711
Ref. (1827) unfed in tab
water for ≥ 2 weeks
∗ TBW
32.0±1.6 7
of lean body mass
Empty urinary bladder
14 C-inulin
Ambystoma gracile
larvae
3210
14 C-inulin
Dicamptodon ensatus
larvae
2310
14 C-inulin
37±1.73
26.4±2.29
27-4011
Ref. (1827) unfed in tab
water for ≥ 2 weeks
Free access to tap water
Empty urinary bladder
1 (1829). 2 (1487). 3 (758). 4 (1193). 5 (1513). 6 (1308). 7 (870). 8 (781). 9 (961). 10 (1764). 11 (1827).
compartment mediating the diffusion of small ions between
cells. Tight junctions between the outermost living cells constitute high-resistance seals (422, 497, 519) of permeability
to Na+ that is slightly above the permeability to Cl− (995,
1261).
Following publication in 1958 of the frog skin model
(956), it was discussed that the electrical potential step at the
apical membrane is positive. This would be expected if the
Na+ conductance of the apical plasma membrane is much
larger than that of K+ of the basolateral plasma membrane.
Subsequent well-controlled cell impalements with microelectrodes showed that the cell is electrically negative with respect
to the outside bath, indicating that the K+ conductance of the
basolateral plasma membrane is much larger than the apical
Na+ conductance (709, 729, 1274). This implies that the apical plasma membrane potential constitutes an additional and
Volume 4, April 2014
major driving force for Na+ uptake across the outward facing
membrane.
Apical Na+ channels: ENaC The apical Na+ channel of
the outward-facing membrane is blocked by a concentration
of amiloride in the low micro-molar range (1067, 1897). The
channel’s molecular phenotype as exemplified by its selectivity, rectification, single channel conductance and amiloride
sensitivity indicated that it belongs to the large family of
epithelial sodium ion channels denoted ENaCs. This has been
verified in a more recent study of cloning and functional characterization of the amiloride inhibitable sodium channel of
the skin of adult R. catesbeiana (849). The uptake of Na+
is stimulated by the amphibian neurohypophyseal hormone,
arginine vasotocin (AVT) (93) and by aldosterone (1276),
which also stimulates the basolateral conductance (1279). It
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Osmoregulation and Excretion
Comprehensive Physiology
Cornified layer
Apical membrane
of first living cell layer
MR cell
Principal cell
compartment
Gland duct
20 μm
Lumen of
mucosal
gland
Basal lamina
Acinar epithelium
Iridiophore
Figure 20
Anatomy of anuran skin (Rana esculenta) with two ion and water transporting
units. The multilayered, heterocellular epidermis is an absorbing epithelium of principal
cells and MR cells. The acinar epithelium of the subepidermal mucous glands is secretory.
Courtesy Dr. Åse Jespersen (993).
is a halmark of the Na+ channel of the anuran skin, that the
open probability is under fast control by the external Na+
concentration via an external Na+ -specific binding site (562,
1899). The Michaelis-Menten-like saturation kinetics of the
unidirectional Na+ influx (128, 498, 938) is due to this mechanism and not to saturating carrier transport with which it is
easily mistaken. It is conceivable that this mechanism prevents
swelling of the epithelial cells when external [Na+ ] suddenly
changes from the low concentration of freshwater to the much
higher concentration of Ringer’s solution of signifiance when
the animal is on land and covered by a near-isotonic surface
fluid produced by the subepidermal glands (see below). Independent of this regulation of the apical sodium permeability
a
), Harvey and Kernan provided evidence for a negative
(PNa
a
by the intracellular sodium ion activfeedback control of PNa
ity (710).
Apical K+ channels The apical K+ channels shown in
Fig. 21A constitute a route for cutaneous elimination of K+
in animals experiencing a body load of potassium (552, 1278,
1310, 1900). This pathway, exhibiting single filing (504), is
normally downregulated, rendering the apical membrane of
principal cells Na+ selective as suggested in the original frog
skin model.
Apical Cl− channels are missing The apical membrane
of principal cells is tight to Cl− even after activation of the
transepithelial passive Cl− permeability (1271, 2001, 2002),
leaving the Cl− uptake to other pathways.
466
Localization and functions of the ‘sodium pump’
The active uptake of Na+ is fueled by the ouabain-sensitive,
rheogenic 3Na+ /2K+ P-ATPase (344, 503, 709, 1275, 1312),
which is expressed exclusively in the plasma membranes lining the lateral intercellular spaces (518, 1227, 1229). The
pump maintains the intracellular Na+ concentration below
and the intracellular K+ concentration above thermodynamic equilibrium (580, 709, 1008, 1277, 1313, 1545, 1774)
(see Table 8). The intracellular [Cl− ] is also above thermodynamic equilibrium (129, 616, 709, 1277, 1545, 2001),
which seems to be maintained by a basolateral Na+ -2Cl− K+ cotransporter in parallel with a low-conductance passive
basolateral Cl− permeability (469, 470, 1890). These mechanisms play a role in cell water volume regulation in frog
skin principal cells (528, 1890, 1891) and in body cells more
generally (287, 783).
K+ channels at the basolateral membranes The rel-
atively high K+ permeability of the basolateral plasma membrane results in an electrical conductance that is several times
larger than that of the apical plasma membrane, and an intracellular electrical potential that is negative with respect to both
the outside bath and the interstitial fluid (709, 729, 1274). The
membrane’s ion conductance has been characterized by noise
analysis and ion channel inhibitors (765, 1898). Intracellular pH, conceivably controlled by basolateral Na+ /H+ —and
Cl− /HCO3 − exchange mechanisms (Fig. 21A), regulates the
open/close kinetics of the apical Na+ —and the basolateral
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Comprehensive Physiology
Osmoregulation and Excretion
(A)
(C)
Outside
bath
(–)
AQP h2/h3
(K+
ENaC
Interstitial
fluid
(+)
Principal cell
compartment
H2O
AQP h3BL
H2O
)
Na+
2Cl–
K+
K+
Na+
(B)
ENaC
Cl–
Na
ENaC
K+
H+
H+ ATP
HCO3–
Na+
ATP
Cl–
H+
Cl–
K+
Na+
Na+
ATP
PKA
c.s
cAMP
ATP
ATP
CA
CO2 + H2O
Cl–
K+
CO2 + H2O
CA
HCO3–
α-Type mitochondria-rich
cell
(D)
+
ATP
Na+
K
+
Cl–
ATP
HCO3–
ATP
Na+
K+
+
Cl–
Na+)
Cl–
+
Interstitial
fluid
(+)
Na+
Cl–
(K+
Outside
bath
(–)
β-Adrenergic
receptor
ATP
HCO3–
Cl–
Cl–
K+
CO2 + H2O
CA
γ-Type mitochondria-rich
cell
H+
Cl–
K+
β-Type mitochondria-rich
cell
Figure 21 Models of the functional organization of the heterocellular anuran skin epithelium. Experimental
approaches and methods are detailed in the text. (A) The large Na+ -transporting syncytial compartment. Cl−
channels are absent in the apical membrane, which contains Na+ channels (ENaC) and K+ channels. Notably,
the Na+ /K+ pumps are located exclusively in the plasma membranes lining the lateral intercellular spaces. (B) The
γ -type MR cell specialized for active and passive Cl− uptake as has been studied in frogs in freshwater and on
land covered by saline of regulated composition, respectively. Shown at the apical plasma membrane are voltageand concentration-gated Cl− channel with its regulatory external Cl− binding site, and the hormone-gated CFTR
like Cl− channel. PKA: protein kinase A; c.s.: catalytic subunit; CA: carbonic anhydrase. As discussed in the text, a
number of observations on acid-base transport and active Cl− uptake are not compatible with the hypothetical γ type MR cell. They are, however, reconciled with two other types of MR cells (α and β), originally discovered in turtle
urinary bladder. (C) Accepted model of an acid-secreting α-type MR cell. (D) Accepted model of a base-secreting
β-type MR cell with associated non-rheogenic active Cl− uptake. Modified from (993).
K+ channels (706, 711, 1886), supposed to regulate the intracellular cation pools when the animal is facing environmental
conditions of varying external Na+ concentrations. Another
function of basolateral K+ channels is to maintain a sufficient
electrical driving force for the passive Na+ uptake through the
apical membrane’s Na+ channels (1272), even at an external
Na+ activity as low as 100 μM, that is, well below that of
many freshwater pools (709). Thus, the relatively high and
regulated basolateral K+ permeability is of significance both
for maintaining intracellular ion pools and cell volume, and for
maintaining whole body extracellular Na+ balance. It is likely
that different types of K+ channels control these functions.
However, they have not yet been identified genetically, which
contrasts the fairly detailed description of their biophysical
properties that include single-filing (345), anomalous rectification (1885), and ATP dependency (1887).
Volume 4, April 2014
Water channels Another important function of the principal cell compartment is to regulate transcutaneous uptake of
water (761, 1126, 1738). This function is mediated by insertion of aquaporin isoforms, AQP-h2 and AQP-h3, into the
apical plasma membrane, which has been studied by AVTstimulation via V2 receptors (14, 712, 1806), isoproterenol
stimulation via β-adrenergic receptors, and hydrin-1, and -2
stimulation via V2 receptors (1364). Water passes the basolateral plasma membrane through constitutively expressed aquaporins characterized as AQP-h3BL (712, 1363, 1791).
MR cells
Mitochondria-rich or mitochondrion-rich (MR) cells display
two types of plasma membrane ion pumps: the P-type Na+ /K+
pump and the V-type H+ pump. Anuran skin has the capacity
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Osmoregulation and Excretion
Comprehensive Physiology
Role of the H+ ATPase of MR cells in energizing Na+
uptake in freshwater The electrical potential difference
+
provides a major driving force for Na uptake across the
outward-facing membrane, which secures uptake of Na+ also
at reduced external Na+ concentrations. Ehrenfeld, GarciaRomeu and Harvey (482) reasoned that at very low external
Na+ concentrations the driving force for Na+ transport across
the apical membrane would reverse, because the membrane
potential would no more overcome the chemical potential
difference across this membrane. They hypothesized that in
vivo the rheogenic H+ pump by hyperpolarizing the apical
membrane energizes the cellular uptake of Na+ from diluted
solutions (482, 708). Following their localization of the proton
pump to MR cells, they modified the hypothesis by pointing
out that a similar effect would be obtained at open circuit
conditions by the external current loop carried by the H+ flux
through MR-cells and the Na+ flux through principal cells
(483, 707).
The H+ ATPase of the γ -type MR cell energizes
active transcellular Cl− transport In the late 1930s,
August Krogh discovered a powerful active Cl− uptake mechanism in the skin of R. esculenta (973, 978) subsequently
found in other species as well (24, 583, 871). It displays halfmaximum saturation concentration in the range of 100 μM to
500 μM of external Cl− (20, 24, 213, 581, 1261). Ehrenfeld
and Garcia-Romeu (480) showed that Cl− uptake at low concentrations is dependent on and tightly coupled to excretion
of HCO3 − (see Fig. 22).
The exchange mechanism is independent of the transepithelial potential difference (480, 848, 970), exhibits cis side
interaction between 36 Cl− and ordinary chloride (848) and is
inhibited by carbonic anhydrase inhibitors (20, 213, 480, 583).
The enzyme is expressed exclusively in MR cells (1578) like
an apical band-3-related protein (421, 895). Taken together,
these findings provide compelling evidence for an apical
Cl− /HCO3 − exchange of MR cells governing Cl− uptake in
freshwater. Several studies have shown that the active Cl−
flux is rheogenic (111, 213, 1173, 2064). It was hypothesized, therefore, that the active uptake of Cl− (and of Br−
468
–300
Jnet,base (neq ⋅ h–1 ⋅ cm–2)
to acidify the external bath by a H+ pump (482, 492, 1119),
which by immunocytochemical labeling was identified as a
V-type H+ pump in the apical plasma membrane of MR cells
in the skin of the semiaquatic frog, R. esculenta (483, 945).
A similar pump was subsequently identified in the skin of the
terrestrial Bufo bufo (847). The pump that seems to correlate
with the previously identified rod-shaped intramembranous
particles of MR cells (207) translocates one positive charge
outwardly for each proton excreted (708, 846). There is evidence that proton pumps are expressed in three types of MR
cells of anuran skin that serve different functions. This issue
is dealt with first. Subsequently, the function of the Na+ /K+
pump of MR cells is discussed. The passive Cl− transport is
also mediated by this minority cell type, which is discussed
toward the end of the paragraph on MR cells.
–200
–100
0
0
100
200
300
400
Jin,Cl (neq ⋅ h–1 ⋅ cm–2)
Relationship between Cl− influx (Jin,Cl ) and net base
excretion (Jnet,base ) in short-circuited frog skin (R. esculenta) exposed
to 2 mM Cl− on the outside and SO4 2− on the inside. Active Na+ flux
is blocked by amiloride. The regression line is drawn according to
Figure 22
Jnet,base = −(0.50 ± 0.06) · Jin,Cl − (16.7 ± 9.3),
r = 0.78 ± 0.15, P < 0.001.
The slope of ∼0.5 is compatible with the finding that the anion
exchange mechanism also mediates Cl− self-exchange. Adapted from
ref. (480).
and I− as well) is energized by the proton pump of the
γ -type MR cell shown in Fig. 21B, which is depicted with
both the Cl− /HCO3 − exchanger and the V-type H+ ATPase in
the apical membrane (1008). Further support for this hypothesis comes from studies of toad skin (B. bufo) demonstrating
that replacement of external Cl− by a non-permeating anion
reversibly acidified the external bath as predicted by the model
of Fig. 21B (492, 846, 1008). In skins of salt-depleted anurans (R. esculenta and B. bufo) bathed with low NaCl on
the outside, the H+ efflux and the Cl− influx were of similar magnitude. Both fluxes were reduced by about the same
amount by the V-type H+ pump inhibitor concanamycin A,
confirming that proton pumps energize active uptake of Cl−
(848).
With both the H+ pump ATPase and the Cl− /HCO3 −
exchanger located at the apical membrane the functional organization of this anuran MR cell-type are clearly different from
MR cells serving whole-body acid/base regulation. Thus, it
has been denoted the γ -type MR cell. As discussed in detail
below, the apical membrane of the γ -MR cell is also the site
of chloride channels that are activated when the frog is on
land and covered by a cutaneous surface fluid generated by
submucosal glands.
Role of the H+ ATPases of α- and β-type MR cells in
eliminating acid and base loads The H+ pump ATPase
energizes cutaneous elimination of protons in acid-loaded
frogs (R. esculenta), which display an increased density of
cutaneous MR cells and increased number of H+ pumps
inserted into the apical plasma membrane by exocytosis in
response to acid loading (707). Aldosterone stimulates the
active proton secretion and a simultaneous uptake of Na+ via
Volume 4, April 2014
Comprehensive Physiology
ENaC, which secures electroneutral transepithelial transport
in open-circuit conditions. Because H+ secretion into the outside bath was associated with base secretion into the inside
bath (465), it is conceivable that this function is mediated by
an α-type MR cell depicted in Fig. 21C, originally discovered in turtle urinary bladder (1759) and now also studied
in epithelia of other lower vertebrates (for further discussion,
see section above on Agnata and Pisces). This hypothesis conforms to the early observation that in frog skin (R. ridibunda),
external acidification was unchanged if sulphate was replaced
by chloride on the corneal side of the skin (493), confirming
that a Cl− /HCO3 − exchange mechanism is expressed at the
basolateral rather than apical plasma membrane. There is evidence that toad skin also contains α-MR cells. Firstly, double
immunostaining showed expression of apical proton pumps
and basolateral AE1/AE2 Cl− /HCO3 − exchangers in flaskshaped MR cells in the skin of B. marinus (206). Secondly, in
9 out of 42 preparations of B. bufo, acidification of the outside bath was independent of the outside anion. Throughout
the year, the toads were kept in a simulated terrestrial habitat
at room temperature with free access to a pool of tapwater
and fed mealworms twice a week. Thus, it is worth noting
that seven of the nine preparations were studied during a onemonth midsummer period, which would indicate seasonal
variation of the functions of toad skin MR cells (846).
MR cells with H+ pumps in the basolateral membrane
and Cl− /HCO3 − exchangers in the apical membrane are configured for active non-rheogenic uptake of Cl− (conf. Fig.
21D). This so-called β-type MR cell serves base secretion in
distal renal epithelia (1759, 1919). Base-loaded frogs excrete
HCO3 − via the skin (1903), and the classical study by GarciaRomeu and coworkers indicated non-rheogenic active uptake
of Cl− in exchange for base in frogs selectively depleted of
Cl− (583). These observations would be in agreement with the
hypothesis that anuran skin under some conditions contains
β-type MR cells.
Putative function of active Na+ transport by MR cells
MR cells display apical amiloride sensitive Na+ channels
and basolateral Na+ /K+ -pumps and are thus configured for
transporting Na+ actively into the interstitial fluid (707, 1007,
1542). With Ringer’s solution on the outside, the flux amounts
to a very small fraction of the active Na+ flux via principal cells (1007, 1542). It is not clarified, however, whether
the uptake of Na+ by MR-cells is of physiological significance in highly diluted solutions. Regarding this question, it
is interesting that the “saturation kinetics” of Na+ uptake in
anurans at Na+ bath < 3 mM exhibits a K1/2 of 200-300 μM
(481, 647, 932, 939). Similar low K1/2 values were observed in
the caecilians, Typhlonectes compressicauda and Ichtyophis
kohtaoensis (1765). The nature of the Na+ bath -dependence of
the Na+ uptake in highly diluted solutions deserves further
studies.
Passive Cl− transport by the γ -type MR cell Experimental studies applying different methods have indicated that
Volume 4, April 2014
Osmoregulation and Excretion
(A) PCl (10–6 cm/s)
(B)
20
GT (mS ⋅ cm–2)
Cl– outside
10
1.0
0.5
Cl– channel
activation
SO42– outside
0
0
40
80
[Cl–]o (mM)
120
–100
0 50
VT (mV)
Studies by Larsen et al. of the dynamic apical Cl− channels of MR cells of anuran skin. (A) Dependence of the permeability, PCl , on Cl− concentration of outside bath at constant VT (ψ o −
ψ i ) = -80 mV (inwardly directed electrical force on Cl− ); as Cl− o
approaches freshwater concentrations, the channels close reversibly.
Adapted from (699). (B) Dependence of the Cl− conductance on VT
at constant [Cl− ]o = 110 mM. GCl is activated in the physiological
range of transepithelial potentials—that is, Cl− channels open when
the driving force on Cl− is in the inward direction (VT < 0 mV) and close
when the driving force is outwardly directed (VT > 0 mV). Adapted from
(995).
Figure 23
MR cells constitute the physiological pathway for passive
transepithelial uptake of Cl− in anuran skin (546, 901, 996,
1007, 1280, 1917, 2001). The passive Cl− conductance (GCl )
of MR cells in the skin of frogs and toads is deactivated in the
absence of Cl− on the outside (956) and reversibly activated
at external Cl− concentrations above 3-5 mM (24, 699, 940)
(see Fig. 23A). This GCl , which is controlled by external Cl−
via an apical chloride-binding site as indicated in Fig. 21B,
has poor anion selectivity. The preference for Cl− above other
anions has been ascribed to an external binding site with a high
Cl− specificity, which allows the channel to open at external
Cl− o above ∼5 mM (699, 971), see ref. (993) for detailed
discussion. In the skin of toads, B. bufo (994, 995), B. viridis
(897) and B. marinus (985, 1007), for Cl− o > 5 mM, GCl is
also potential dependent in such a way that depolarization of
the apical plasma membrane of MR cells activates GCl (994)
in the Ussing chamber accomplished by hyperpolarization of
the skin potential (995). This seems different from the skin of
frogs (e.g., R. esculenta, R. temporaria and R. pipiens), which
does not always show a similar VT dependence of GCl unless
prior stimulated by theophylline, cAMP or forskolin added to
the serosal bath (972, 1583). The Cl− conductance is slowly
activated (30-180 s) following a hyperpolarizing step of VT
(= ψ outside bath − ψ inside bath ), and deactivated at VT > 0 mV
(897, 900, 903, 985, 995, 1003, 1583, 2000, 2001). As a result,
the steady state GCl depicts an inverted S-shaped function of
VT (see Fig. 23B).
In voltage clamp experiments, the Cl− uptake across the
isolated skin epithelium is associated with volume expansion
of MR cells (546, 1007) of a time course similar to that of
the transepithelial Cl− current activation (1007). At fully activated GCl , the Cl− current through single MR cells is quite
impressive, ranging from about −1 to about −15 nA (546,
996, 1280). Noise analysis of whole cell Cl− currents indicated a large single channel conductance of about 250 pS
469
Osmoregulation and Excretion
Comprehensive Physiology
(994), which was indicated also in single-channel recordings
of isolated MR cells (1732). The passive Cl− permeability
of MR cells is controlled also via β-adrenergic receptors at
the basolateral membrane and stimulated by forskolin and
membrane permeable cAMP analogues (405, 898, 902, 972,
1282, 1283, 1582, 2002). In toad skin, this eliminated the
time and potential dependence of GCl within the physiological range of transepithelial potentials (1582, 2002), associated
with the activation of a small (8-10 pS) membrane-potentialindependent CFTR-like Cl− channel of the apical membrane
(1732). As mentioned above, in studies of the skin of R. esculenta, R. temporaria and R. pipiens, similar treatments were
reported often to be a prerequisite for GCl being activated by
hyperpolarizing VT (972, 1583). The γ -MR-cell shown in Fig.
21B depicts these functions as governed by two different apical Cl− channels in agreement with whole cell noise analysis
(994) and single channel recordings (1732). As an alternative
and still possible interpretation, Katz, Nagel, and Rozman
hypothesized that phosphorylation and potential stimulation
target a common apical Cl− pathway (1281, 1583). In line with
this hypothesis, it has been discussed that large-conductance
Cl− channels are composed of small Cl− channels of coordinated activity (2000). This interpretation would be compatible with observed single-channel iCl /V-relationships of
the big Cl− channel that did not exhibit a common GHKpermeability, but a range from 0.3·10−12 cm3 /s to 1.18·10−12
cm3 /s (1732).
reuptake of NaCl (and water) supposedly constitutes a major
function of the epidermal cells. In freshwater, closure of the
epidermal Cl− channels prevents cutaneous loss of Cl− to the
environment so that not only Na+ but also Cl− is taken up
by active transport. The interrelationship between these functions has been studied experimentally by flux-ratio analysis
of unidirectional Cl− fluxes (e.g., 990). For Cl− at 20 ◦ C,
Eq. 12d is
log10
in
JCl
z Cl (VT − E Cl )
out =
JCl
58.0
(14)
Figure 24 shows the relationship between the measured flux
ratio and the external driving force, zCl ·(VT − ECl ), which is
positive in the inward direction. The theoretical flux ratio is
indicated by the straight line of slope, 10−3 ·F·log10 e/(R·T) =
1/58 mV−1 . For an inwardly directed driving force, Cl− channels are open, allowing for a passive flow of Cl− from bath
to interstitial fluid (see the right-hand side of the diagram).
For VT > 0 mV, the Cl− channels close, revealing the active
component of the transepithelial Cl− flux (open symbols).
This component is seen also when external Cl− is reduced
to freshwater concentrations (conf. the closed symbol of
Fig. 24). With the apical Cl− channels closed, in freshwater,
100
Dynamic coupling of active Na+ uptake by
principal cells and passive Cl− uptake by MR cells
[Cl–]o = 112 mM
[Cl–]o = 3.3 mM,
−
Passive and active Cl− transport—the frog on land
and in freshwater
The concentration and voltage dependence of the passive Cl−
permeability discussed above seem to be an adaptation of
amphibians to a life alternating between freshwater and land
(993). On land, where the skin is covered by a thin layer of
saline produced by subepidermal exocrine glands (see below),
470
ECl = –82 mV
10
IN
OUT
Flux ratio (JCl /JCl )
At open-circuit conditions where VT governs passive Cl
uptake via MR cells, the rheogenic active flux of Na+ and the
simultaneous electrodiffusion of Cl− constitute a current loop
across the epithelium. It follows that stimulation of the active
Na+ flux through principal cells—for example, by increasing
the number of open ENaC channels—will increase the current through MR cells, leading to an ohmic depolarization of
their apical plasma membrane that in turn activates Cl− channels. The range of VT in which the Cl− permeability is taken
from deactivated to fully activated state (Fig. 23B) is similar
to the voltage range generated by the active Na+ flux when
the apical Na+ permeability of principal cells is passing from
non-activated to fully activated state. Quantitative analysis
showed that this type of regulation results in tight coupling
of the active Na+ flux by principal cells and the passive Cl−
flux through MR cells (993, 1004).
1
–100
–50
0
50
–(VT – ECl) (mV)
100
Flux-ratio analysis of Cl− transport across the isolated
skin of B. bufo. The straight line was calculated by Eq. 14 in the text.
The graph shows that the experimental flux ratio follows the theoretical
line for electrodiffusion if the driving force, zCl ·(VT - ECl ), is in the inward
direction at elevated external Cl− , where the apical Cl− channels of
the γ -MR cells are activated (right-hand side). If zCl ·(VT − ECl ) is in the
outward direction (left hand side), the apical Cl− channels are closed
while the active Cl− fluxes, fueled by the apical H+ V-ATPase, and the
Cl− :Cl− exchange diffusion fluxes become the dominating modes of
Cl− transport. Thus, the Cl− flux is always inwardly directed, whether
the animal is in freshwater of low Cl− or on land and covered by a
cutaneous surface fluid of high Cl− concentration (993).
Figure 24
Volume 4, April 2014
Comprehensive Physiology
Osmoregulation and Excretion
active uptake of Cl− is the dominating transport mode, as
found by August Krogh in 1937 (978).
active Cl− transport is fueled by ATP hydrolysis at the lateral
Na+ /K+ -pumps (143, 1226, 1231). A lumen-negative electrical potential and solvent drag drive the paracellular Na+ flux.
An active K+ secretion is accomplished by the lateral Na+ /K+
pumps in series with maxi-K+ channels, which by singlechannel recordings were shown to be co-expressed with CFTR
at the luminal plasma membrane (1735) as depicted in Fig. 25.
During fluid secretion, the major fraction of K+ , which is
transported into the cells by the Na+ /K+ pump and the Na+ 2Cl− -K+ cotransporter, is recirculated into the interstitial fluid
by maxi-K+ channels at the contra-luminal membrane (28).
Characterized by immunofluorescence labeling, the luminal
plasma membrane is also the seat of AQP-x5-like aquaporins
(764). In the above studies, glandular secretions were elicited
via α 1 -adrenergic, β-adrenergic, muscarinic cholinergic or
prostaglandin E2 receptors. As discussed below, gland secretions serve a number of different functions, but their coupling
to the above signaling pathways is unknown.
Subepidermal Glands
The density of glands (see Fig. 20) is in the order of 103 per
cm2 . Mucous glands are 5-10 times more abundant than granular glands and scattered throughout the entire surface of the
body, while granular glands are relatively more abundant on
the back and with a tendency of being aggregated in the neck,
head, and tail regions. They are both innervated by adrenergic fibers, and secretion is stimulated by adrenergic and
cholinergic agonists and humoral substances (143, 673, 957,
1226, 1309, 1699, 1733, 1892). The mucous-gland acini/skin
surface-area ratio varies between 0.1 and 2.4, with the larger
ratios observed in species from arid habitats (1034-1037).
Ion pathways and water channels in frog
skin glands
Ion composition and rate of fluid secretion
Similar to other vertebrate exocrine glands, the ion secretion by frog skin is driven by active Cl− transport (957).
Chloride secretion by the electrically coupled acinar cells
(1733) is brought about by a Na+ -2Cl− -K+ cotransporter in
the basolateral membrane (143, 1226, 1231) and Cl− channels
in the luminal plasma membrane that are cAMP- (494, 1734)
and Ca2+ activated (673, 1733) (see Fig. 25). The secondary
M1/M3 α1-adr
β-adr
EP2/EP4
G
Conductance measurements of the outflow from nervestimulated frog skin glands (R. temporaria) indicated that
the secreted fluid is composed of about equal amounts of
Na+ and Cl− (989). Table 10 lists the ion composition of
hormone-stimulated secretions of R. pipiens and R. esculenta. The in vivo Na+ and Cl− concentrations are a little
AC
Interstitial
fluid
K+ 2Cl–
Na+
G
+
ATP
IP3
cAMP
K+
K+
ATP
Ca2+
Slow
Na+
Gap junctions/
connexins
Fast
+
Cl–
K+
+
Cl–
?
+
Na+
+
Tight junction
?
CFTR
Na+
Lumen
Figure 25
Ion pathways of an acinus cell of submucosal glands of frog skin. The rate
of secretion is controlled by the activity of luminal Cl− channels, which are regulated
by cAMP and intracellular Ca2+ . The luminal K+ channels are Ca2+ and depolarization
activated and responsible for the relatively high K+ of the secreted fluid. The Na+
channels may be of importance for regulating the tonicity of the primary secretion.
The model compiles results of pre-steady state flux-ratio analyses (1892), whole-cell
and single-channel patch clamp studies (1733-1736) and investigations of signaling
pathways with fluorescent imaging techniques (673). Modified from (1733).
Volume 4, April 2014
471
Osmoregulation and Excretion
Table 10
Comprehensive Physiology
Rate of Production and Composition of Mucous Gland Secretions
Frog in vivo (R. pipiens)
[Na+ ]
[Cl− ]
HCO3 −
[K+ ]
Stimulus
Secretion rate
ml·h−1
1 Control
0.02±0.01
–
–
–
–
–
1 Noradrenaline,
0.22±0.07
82.8±2.4
64.9±1.7
19.1±1.4
14.3±1.0
7.89±0.08
1 Aminohylline,
2.5 mg
0.36±0.04
98.0±7.5
64.0±1.5
16.8±1.8
27.5±1.6
8.68±0.06
1 Aminophylline,
5.0 mg
0.53±0.08
90.6±5.6
69.8±6.0
16.0±0.9
31.8±2.5
8.72±0.07
1 Blood
mmol/l
pH
400 μg
–
104.3
75.7
3.6
28.3
7.47
2 Control
plasma
0.16±0.04
26.5±4.6
27.0±6.1
23.5±2.8
3.5±0.4
7.56±0.04
2 Adrenaline,
0.34±0.02
95.4±4.4
61.9±2.0
16.8±0.8
18.4±2.5
7.65±0.06
–
104.8
77.0
4.6
32.0
7.47
1 mg
2 Blood
plasma
Isolated short-circuited skin, amiloride outside (R. esculenta)3
JNa
Stimulus
Secretion rate
μl·h−1 ·cm−2
JCl
JK
[Na+ ]
nmol·h−1 ·cm−2
[Cl− ]
[K+ ]
mmol/l
Sum mosmol/l
PGE2 , 2 μM
2.16±0.49
130±20
289±45
33±3.6
60.3±9.2
134±21
15.3±1.7
209
+ theophylline, 1 mM
5.51±1.04
338±32.4
606±54
69±11
61.4±5.9
110±9.8
12.5±2.0
184
Serosal fluid
–
115
118
2.5
240
1 (1934). 2 (247). 3 (143).
lower than the concentrations of the extracellular fluid while
that of K+ is significantly above extracellular K+ , which are
characteristics also of the gland secretion by the isolated
skin and of primary secretions of other vertebrate exocrine
glands. A major difference is the relatively high concentration of Cl− in vitro (Table 10), which is due to the isolated
skin being short-circuited; Cl− was carrying the major fraction of the short-circuit current, which matched the electrical
charge flux carried by the simultaneously measured fluxes
of Na+ , K+ and Cl− . The advantage of this method is that
the ion flow (the short-circuit current with epidermal ENaC
blocked by amiloride) and the fluid movement are measured
simultaneously with good time resolution. Initially, stimulation elicits large transient current and fluid flows followed by
smaller steady-state flows maintained for several hours (143).
The in vivo studies of Table 10 with mean secretion rates
ranging from 0.22±0.07 to 0.53±0.08 ml·h−1 were obtained
in R. pipiens weighing 30-35 g (247). The surface area of
472
similar-sized R. esculenta is about 133 cm2 (975). Using this
number, in vitro rates listed in Table 10 correspond to wholebody rates of 0.29±0.07 and 0.73±0.14 ml·h−1 , respectively,
which are comparable with rates measured in vivo.
Function of skin gland secretions
Besides diffusible electrolytes, the mucous gland secretion
may contain glycosylated mucins and mucopolysaccharides.
The bioactive material produced by the granular glands consists of neuropeptides, antimicrobial peptides, lysosomes, and
antibodies, which are spread over the body surface in the
cutaneous surface fluid (CSF). By covering the surface of the
animal, the secretion keeps the skin moist, which is important for skin respiration and for preventing desiccation of the
epidermal cells. CSF also protects against bacterial infection
and entry of molds, and for making the body slippery, which
helps the animal escape from predators (246, 456, 1054, 1570,
Volume 4, April 2014
Comprehensive Physiology
Osmoregulation and Excretion
1639, 1756, 1918). Due to a particular abundance of mucous
glands in the abdominal seat patch of terrestrial toads, Hillyard (764) hypothesized that mucous glands of this region are
of significance for validating the quality of a hydration source
prior to cutaneous drinking. It has been suggested that gland
secretion in some species serves evaporative cooling (180,
235, 592, 905, 1052, 1057, 1676).
The hormone-stimulated gland secretion is nearisoosmotic or hypoosmotic with the body fluids (Table 10).
Thus, if the cutaneous surface layer is maintained by gland
secretion, the diffusion flux of water across the epidermis
cannot be in the direction from the body fluids to the surface of the skin (993). A recent study verified this notion by
measuring directly the osmolality and the Na+ and K+ concentrations of CSF and the lymph in R. esculenta subjected
to raised environmental temperature or isoproterenol injection (1001, 1002). The osmolality of CSF sampled in vivo
was 181±8 mosmol/kg at 30-34◦ C and 191±9 mosmol/kg
in isoproterenol injected frogs at 15-23◦ C, while the average
osmolality of the lymph was 249±10 mosmol/kg (see Fig. 26).
Beyond being governed by the transepithelial osmotic gradient, the inward flow of water is coupled with the inwardactive Na+ flux (1313, 1314), probably driven by a slightly
hyperosmotic paracellular compartment (1009). Therefore, in
amphibians covered by a cutaneous surface fluid, the source
of EWL is the fluid secreted by the mucous glands, and not
water diffusing through the skin as hitherto assumed. This
physiological mechanism coping with the unavoidable evaporation in amphibians prevents desiccation of the epidermal
300
EWL at 30–34 °C
200
osm
CCSF (mosmol/Kg)
100
n=6
n=7
n=4
n=8
n = 12
E1
E2
E3
E4
E5
0
300
Isoproterenol stimulated
200
100
n=4
0
I1
n=8
23 °C
I2
n=8
I3
n = 16
15 °C
I4
Frog #
Figure 26
Osmolality of the cutaneous surface fluid (CSF) of R. esculenta (1001, 1002). Number of samples indicated by n is shown
for each animal analyzed. Above: CSF samples from animals during evaporative water loss (EWL) at 30-34◦ C ranged from 101 to
253 mosmol/kg (mean ± s.e.m., 181±8 mosmol/kg). Below: The
osmolality of CSF samples from isoproterenol-treated frogs at 15
and 23◦ C ranged from 123 to 281 mosmol/kg (mean±sem, 191±9
mosmol/kg). Horizontal bars indicate lymph concentrations of 10
frogs, 249±10 mosmol/kg (range, 228-330 mosmol/kg).
Volume 4, April 2014
cells. Paragraphs below discuss further adaptations to terrestrial environments, such as morphological skin specializations
and behavior of importance for reducing water loss by evaporation.
Turnover of the Cutaneous Surface Fluid:
Functional Coupling of Gland Secretion and
Epidermal Ion Uptake
If ions were not reabsorbed via the epidermal epithelium,
evaporation of CSF would lead to an increase in its osmolality.
In a study of R. esculenta of an average body mass of 43 g and
a mean body surface area of 117 cm2 , Rey observed a water
loss 570 mg·h−1 or 1.35 nl·cm−2 ·s−1 by evaporation (1529).
With a NaCl concentration of CSF ≤ 100 mM, a CSF height
of 8 μm and no reabsorption of ions, after no more 8 min the
NaCl concentration would approach 527 mM, corresponding to a nominal osmolarity of 1054 mosmol/l. The estimate
depends on the height of CSF tentatively assumed similar to
the airway surface fluid of about 8 μm (1731, 1808, 1987).
This might be an underestimation since the outer cornified
layer of frog and toad skin is freely equilibrating with the outside medium (1543, 1545). If, on the other hand, the cornified
layer confines the thickness of CSF, it would be no more than
about 5 μm. Independent of the height of CSF, in the above
example maintenance of a near-isotonic concentration of 100
mM NaCl would require absorption fluxes of Na+ and Cl−
of 135-pmol·cm−2 ·s−1 . Numerous studies have shown that
fluxes of this magnitude of Na+ by the principal cells (active
transport) and of Cl− by MR cells (passive transport) are in
the lower range of the capacity of the epidermal transport
systems at physiological skin potentials. Figure 27 summarizes the functional coupling of mucous gland secretion and
epidermal reuptake of ions and water.
Regulation of osmolality and volume of cutaneous
surface fluid
The function of CSF discussed above rationalizes the enigmatic finding that the Na+ permeability of the apical plasma
membrane of the principal cells of frog skin is sensitive
to small osmolality differences across the epithelium (202).
Thus, the Na+ uptake was stimulated significantly by increasing the outside osmolality (π o ) by < 10%, while a bilateral
increase keeping the osmolality of the serosal fluid (π serosa )
equal to that of the outside solution (π o = π serosa ) had little if
any effect. If the osmotic gradient was reversed (π o < π serosa ),
the apical Na+ permeability decreased. Similar effects on the
Na+ permeability were obtained if the external osmotic displacement was obtained with NaCl, sucrose, KCl and DMSO,
showing that it is the osmolality gradient that regulates the
apical Na+ permeability, independent of how the gradient is
established. This type of regulation of the active Na+ flux and
the parallel change in the passive flux of Cl− via the electrical gating mechanism discussed above would enable effective
control of the ion content of the small volume of CSF. The
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Osmoregulation and Excretion
Comprehensive Physiology
sensing mechanism(s) and the transduction pathways leading
to the aforementioned activation of ENaC are unknown.
With toad skin epithelium (B. bufo) bathed bilaterally with
Ringer’s solutions of identical composition, β-adrenergic
receptor stimulation resulted in an inward transport of nearisotonic fluid (1314). This is accomplished by cAMP activation of Cl− channels of MR-cells (2002) and ENaCs of
principal cells (87) associated with mobilization of AQP2a
isoform water channels into the apical plasma membrane of
the principal cells (1364). The function of this type of fluid
transport would be to regulate the volume (height) of CSF at
reduced EWL, for example, at low skin temperature and/or
high relative humidity. Thus, during evaporation of water into
the atmosphere, it is conceivable that the volume and composition of CSF are maintained by a balance between fluid
secreted by subepidermal glands and water and ions absorbed
by the surface epithelium, which is inferred also from the
direct measurements of CSF in Fig. 26.
Energy expenditure associated with turnover of
cutaneous surface fluid
With reference to Fig. 27 depicting the secretion-reabsorption
couplings of ions and water in amphibian skin under terrestrial conditions, the energy expenditure associated with
the turnover of CSF can be estimated by considering the
oxygen consumption associated with Na+ absorption by the
epidermal epithelium and Na+ secretion by the glandular
epithelium, respectively. The active transport by the epidermal cells of 18 mol Na+ is associated with an uptake of
1 mol O2 (1025, 2071). This number, which is an average of
mosmol/kg
measurements spanning a large range, is in accordance with
3 mol Na+ transported for 1 mol ATP hydrolyzed by the
3Na+ /2K+ ATPase and a mitochondrial P/O ratio of 6. Owing
to Na+ recirculation in isotonic fluid secretion, the above stoichiometry of 18 Na+ /O2 was estimated to be reduced to 3.4
Na+ /O2 (1892). Returning to Rey’s study (1529) mentioned
above with the above Na+ /O2 ratios, the secretion and the
reabsorption of 135 pmol·cm−2 ·s−1 by the 42.6 g frog of a
surface area of 117 cm2 would require an O2 consumption of
∼22 μmol·h−1 . The O2 uptake of similar sized frogs is about
145 mmol·h−1 (975). Thus, the energy expenditure associated with the turnover of CSF is insignificant compared to the
frog’s total energy metabolism.
Mechanism and physiological significance
of uphill water transport by the epidermal
epithelium
Skin preparations bathed bilaterally with Ringer’s solution
transport water in the inward direction (1517) at a rate given
by the active Na+ flux (1313, 1314) (conf. the section above on
“Biophysical Concepts in Osmoregulation”). Water absorption still prevails if the external fluid is hypertonic provided the
osmolality does not exceed the epithelium’s capacity of uphill
water transport. Thus, the water absorption indicated in Fig. 27
would proceed at moderate hypertonicity of CSF. In the
semi-aquatic R. esculenta, water flow stopped when the outside osmolality was raised by Crev = 15.5±3.0 mosmol/kg
above that of the Ringer’s osmolality (1313), while in the
terrestrial B. bufo, Crev = 28.9±3.9 mosmol/kg (1316).
With the intercellular spaces constituting an osmotic coupling
EWL
CSF
Cornified layer
181 ± 8
Basal
lamina
249 ± 10
Na+ /K+ pump
H2O
Cl–
Na+
Cl– channel
Na+ channel
Figure 27 Proposed functional coupling of ion and water transport by subepidermal
glands and epidermal epithelium of anuran skin; after E.H. Larsen (993). Two MR cells
are shown in grey. On land, the skin is kept moist by the cutaneous surface fluid (CSF)
produced by the mucosal glands. Water evaporates from the surface fluid (EWL), and
the surplus of Na+ is absorbed by principal cells (active transport) and Cl− by γ -MR
cells (passive transport). The volume of CSF is maintained by the balance between
fluid secretion by subepidermal glands and water reabsorption by solute coupled fluid
transport and/or by osmotic water uptake across the surface epithelium. Measured
osmolalities are given for the interstitial fluid (lymph) and for CSF during heat-induced
evaporative water loss.
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Comprehensive Physiology
compartment (1009) and water flow through the epithelial
cells, the capacity of uphill water transport would be governed by the maximal osmolality of the fluid in the maze
of lateral spaces and the water permeability of the plasma
membranes (1006, 1961).
If the rate of evaporation is too fast for the ion uptake
mechanisms to keep pace with the water loss to the atmosphere, the osmolality of CSF exceeds the capacity of uphill
fluid transport, abolishing epidermal reuptake of water.
Osmoregulation and Excretion
and that both of these functions are energized by ATP hydrolysis at Na+ /K+ pumps. Further evidence for rheogenic Na+
reabsorption was obtained in a study of the pronephric duct
of the Japanese black salamander, Hynobius nigrescens, in
which ENaCα was immunolocalized at the luminal membrane
of epithelial cells that expressed the Na+ /K+ ATPases at the
peritubular membrane (1882). A putative NHE3 isoform of
the Na+ /H+ exchanger has been immunolocalized at the luminal membrane of the anterior portion of the pronephric duct
in the pronephros of this species, indicating a luminal uptake
mechanism for Na+ coupled to proton secretion (981).
Kidney: Pronephros
During the amphibian life cycle, two kidney generations are
functional, the larval pronephros and the adult mesonephros,
the latter being functional already during larval stages.
Early studies provided indirect evidence for the pronephros
being a larval osmoregulatory organ that eliminates water
and waste products and reabsorbs substances essential for
metabolism (548). The morphology and ultrastructure of the
larval pronephros are outlined briefly based upon a study of
B. viridis by Møbjerg et al. (1244). Each of the paired
pronephroi consists of an external glomerulus located in the
coelom and a single convoluted tubule, which opens into the
coelom via three nephrostomes (2-5 nephrostomes in urodeles and an even larger number in gymnophiones [548]). The
capillary loops of the glomerulus are covered by an epithelium, which corresponds to the visceral layer of the Bowman’s capsule of the mesonephros. The filtration barrier is
much like that of the adult kidney composed by a fenestrated
endothelium, a three-layered basement membrane, and processes of epithelial podocytes. From the coelom, the primary
urine is swept into three branches of the nephrostome by the
anteriorly located short ciliated segments. The urine continues through three branches of the proximal tubule that merge
into a common proximal tubule followed by a distal tubule
and a nephric duct. The urine from each of the two nephric
ducts passes through the collecting ducts of the mesonephros
before flowing into the cloaca. Intercalated (MR) cells are
lacking in the pronephric duct, but are present in the collecting duct and in the nephric duct in larvae of a body length of
14 mm (B. viridis). A well-developed brush border of the proximal tubules with apical endocytotic apparatus and basolateral
invaginations points to ultrastructural specializations for vectorial transport of ions and water. Functions of the fully differentiated pronephric duct of the A. mexicanum were addressed
recently (715). Impalements with microelectrodes of single
microperfused tubules of larvae stage 46-54 indicated negative intracellular potentials averaging between −75 mV and
−80 mV. Peritubular and luminal ion substitutions revealed
a relatively large basolateral K+ conductance and Na+ and
K+ conductances in the luminal membrane. Immunolabeling
and confocal laser scanning microscopy localized Na+ /K+
ATPases to the highly invaginated basolateral membranes.
Thus, it seems that the pronephric duct is important for diluting the urine formed by ultrafiltration and for excreting K+ ,
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Kidney: Mesonephros
The number of nephrons of each kidney depends on species
with no more than 60 in the small African dwarf frog,
Hymenochirus boettgeri of a body mass of 1.3 g and 8,000 in
large Colorado river toad, B. alvarius of a body mass >200 g
(1884). In B. bufo, the number in each kidney is ∼3,000
(1245), while it is ∼1,700 in the fossorial African caecilian Geotrypetes seraphini (1243). As indicated in Fig. 28,
the nephron of the metamorphosed amphibian consists of a
Malpighian corpuscle (the Bowman’s capsule surrounding a
glomerulus), a ciliated neck segment, a proximal tubule, a ciliated intermediate segment, an early and a late distal tubule,
and a collecting tubule, which opens into a collecting duct
Collecting duct
Collecting
duct system
Collecting tubule
Late distal tubule
Distal
tubule
Distal
nephron
Early distal tubule
Intermediate segment
Proximal tubule Sll
Proximal
nephron
Proximal tubule Sl
Neck segment
Bowman’s
capsule
Glomerulus
Peritoneal
Malpighian
corpuscle
funnel
Figure 28
Generalized diagram of amphibian nephron with
nomenclature indicated. The proximal nephron comprises Malpighian
corpuscle, the ciliated neck segment, the proximal tubule and the ciliated intermediate segment. The subdivision of SI and SII segments is
based on dimension of brush border and basolateral infoldings. The
Distal nephron comprises the early and late distal tubule, the collecting
tubule and the first unbranched portion of the collecting duct system. As
shown here, in urodeles and caecilians, the peritoneal funnel connects
the proximal nephron with the coelom. In anurans, this connection is
lost and the peritoneal funnel opens into the peritubular vessel surrounding the nephron. From (1243).
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Osmoregulation and Excretion
shared with other nephrons. In salamanders and caecilians,
a ciliated peritoneal funnel opening close to the glomerulus
connects the lumen of the nephron with the coelom. In anurans, this connection between the nephron and the coelom is
lost, and the peritoneal funnel opens into the peritubular vessel surrounding the nephron. Renal arteries from aorta supply
blood to the kidneys, which also receive vessels from the renal
venous portal system (257, 314, 1070, 1243, 1245, 1596,
1598, 1883). The anatomical description of distal nephron
segments of different amphibians has resulted in several
synonyms, which are discussed in (1245). Evolution of the
amphibian nephron is considered in (1243).
The literature on renal functions of amphibians is
reviewed in (162, 376, 764, 1670). The discussion below
focuses on two major functions related to the environmental
physiology of amphibians. In freshwater, the kidneys produce a large volume of highly diluted urine for eliminating
the water taken up by osmosis across the skin. In terrestrial
habitats, the kidneys produce a small urine volume. Thus,
even if amphibians cannot concentrate urine, renal water conservation is well developed and after a loss of only a few
percent of body water, they can become anuric. The urine
volume depends on the rate of primary urine production and
the tubular processing of water.
Glomerular filtration
The glomerular filtration rate (GFR) is the major determinant of the rate of urine production (544, 586, 1624), which
depends on the number of active nephrons, the net glomerular
filtration pressure, and physical properties of the filtration barrier, that is, total area, filtration coefficient and fractional filtration rate (1432, 1434). Three studies of single-nephrons of
freshwater-acclimated urodeles and anurans indicated singlenephron filtration rates (SNGFR) between 0.1 μl·h−1 and
1 μl·h−1 (609, 1432, 1922). In a study of Amphiuma kidney,
Persson and Marsh (1433) demonstrated a link between flow
dependent entry of Cl− into the epithelial cells of the early
distal tubule and SNGFR. This observation and results of
previous studies (1434, 1727) suggest that tubulo-glomerular
feedback constitutes an intrarenal mechanism by which blood
flow in afferent arterioles and GFR is regulated by the flow
rate in the distal tubules. The reduced GFR seen upon transfer
from freshwater to either terrestrial conditions (1624, 1669,
1765, 1876) or increased salinity (1192, 1632, 1678) is caused
by a reduction of the number of filtering glomeruli, presumably with little or no contribution of glomeruli that filter
continuously at reduced rate (376). The downregulation of
GFR and the increased tubular water reabsorption upon water
restriction operate fast and are independent of prior desiccation or extracellular volume loss (1435, 1876). The posterior
pituitary ADH decreases GFR and increases tubular water
reabsorption (21, 95, 100, 587, 1614). There are additional
as yet unidentified regulatory mechanisms involved, which is
indicated by the observation that surgical elimination of the
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neurosecretory system that produces the antidiuretic hormone
was without effect on anuran water metabolism (873, 874).
Proximal tubule
The primary urine is an ultrafiltrate of plasma formed by the
fenestrated endothelial cells of the glomerular capillary and
the foot processes of the epithelial podocytes, which constitute the visceral layer of Bowman’s capsule (1938, 1979). The
proximal tubule belongs to the class of “leaky epithelia” with
an intercellular (junctional) resistance, which is between one
and two orders of magnitude less than the transcellular resistance (159, 610). In anurans and salamanders, the transepithelial potential difference (VT ) is lumen negative, varying
numerically from 3 mV to 15 mV governed by the active Na+
flux and a low resistance paracellular ‘shunt’ (106, 160, 608,
1777, 1985). The epithelial cells are furnished with brush border, basolateral infoldings and an apical endocytotic apparatus
(314, 1185, 1243, 1245, 1597). The Na+ /K+ ATPase is abundantly expressed in plasma membranes lining the lateral intercellular spaces and basolateral infoldings (1183-1185). The
major fraction of the ultrafiltrate including glucose (1921),
amino acids (1367) and proteins (1492) is reabsorbed in the
proximal tubule as isotonic fluid coupled to the transport of
Na+ (2013), which is active (1748). Water can flow across the
epithelium through both the cellular (“translateral”) and the
paracellular (“transjunctional”) pathway (1866), as indicated
in Fig. 1B. The major influxes of Na+ and Cl− across the brush
border membrane are mediated by Na+ /H+ - and Cl− /HCO3 −
exchange mechanisms, respectively (155, 1646). In addition
to the Na+ /K+ pump flux, the exit of Na+ across the peritubular membrane takes place as cotransport with HCO3 −
of a stoichiometry estimated to be 3 HCO3 − : 1 Na+ (154,
1095). The exit of one Cl− across the peritubular membrane is
in exchange with one Na+ and two HCO3 − from the peritubular side (677, 1092). In parallel with this mechanism, there is
evidence also for Cl− /HCO3 − exchange that would transport
Cl− into the cell from the interstitial fluid (473, 2060). Further to serving transtubular H+ and HCO3 − fluxes, the above
acid-base transporters are supposed to regulate intracellular
pH (154, 155). In Necturus, a cell negative basolateral membrane potential of about −70 mV is governed by a relatively
large K+ conductance of the peritubular plasma membrane
(608).
Early distal tubule: Diluting segment
Based on electrophysiological criteria, Stoner (1777) identified two different segments of the distal tubule in frogs, toads
and salamanders; an early distal tubule, denoted the diluting
segment of a lumen-positive VT , and a late distal tubule, sometime referred to as the junctional segment, of a lumen-negative
VT . In Ambystoma, the functional heterogeneity could be correlated with morphology and ultrastructure at cell level (768).
As indicated by the name, in the diluting segment ions and
water are separated by reuptake of Na+ and Cl− through the
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Comprehensive Physiology
tubular epithelium of a relatively low water permeability (678,
679, 1777). Thus, it is in this segment and in the collecting
duct system that the osmolality of the tubule fluid is reduced in
freshwater-acclimated animals. The magnitude of the lumenpositive VT seems species dependent, varying from +5 and
+8 mV in B. marinus and Amphiuma means, respectively, to
+22 mV in Triturus alpestris (1433, 1777, 1818). Flux studies and intracellular ion activity measurements in anurans and
salamanders indicated that the uptake mechanism in the luminal plasma membrane is an electroneutral cotransporter of a
stoichiometry of 1 Na+ :1 K+ : 2 Cl− (1351, 1352, 1354-1356,
1777). The lumen-positive VT is due to a luminal membrane
potential (cell negative) that exceeds numerically the potential
difference across the peritubular plasma membrane (1353).
This owes to a relatively large passive K+ conductance of
the luminal plasma membrane, which returns a major fraction of K+ taken up by the cotransporter. In Ambystoma, the
luminal K+ conductance and a Na+ /H+ exchange mechanism were stimulated by K+ loading (1350, 1357). In a study
combining intracellular potential and cell volume recordings,
Guggino (676) identified two cell types of the early distal
tubule of Amphiuma: one cell type in which both Cl− and
K+ move across the peritubular membrane by electrodiffusion, and another cell type in which the two ions exit across
the peritubular membrane in an electroneutral fashion via a
K+ -Cl− cotransporter. In a subsequent review, Guggino et al.
(678) concluded that functionally the amphibian early distal tubule has much in common with the mammalian thick
ascending limb.
Late distal tubule
The functional organization of the late distal tubule has been
more difficult to study because the electrophysiological properties change along the tubule. Following Stoner’s definition,
the late distal tubule is a “tight” epithelium with an upperlimit transepithelial resistance of about 1,200 ·cm2 and a
lumen-negative VT averaging from −6 to (occasionally) about
−40 mV with more negative values recorded toward the collecting tubule (26, 1469, 1777, 1818). Anagnostopoulos and
Planelles (26) measured the electrochemical potential differences of Cl− , K+ , Na+ and H+ across the peritubular and
luminal plasma membranes of Necturus in vivo, which can
be summarized as follows. With the renal surface superperfused by Ringer’s solution of pH = 7.4, the ion activities of
the peritubular capillary blood were (mM), 71.0 Na+ , 2.5 K+
and 70.5 Cl− , pH = 7.37. With the urine stemming from
the diluting segment, the tubule cells of the late distal tubule
maintained activities of the luminal fluid of about (mM) 9
Na+ , 2.5 K+ and 12 Cl− and a pH of 6.5 at an average VT
of −14 mV. For Na+ , K+ and Cl− , the measured transepithelial electrochemical gradients were opposite to the established
inwardly directed net transports, which therefore would have
to be transcellular and active. The steady-state distribution of
protons indicated active secretion of this ion. With an apical membrane potential of about −60 mV (cell negative) and
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Osmoregulation and Excretion
the following intracellular activities (mM), 9.0 Na+ , 65.8 K+
and 5.5 Cl− , the active step for the Cl− flux would be at
the luminal plasma membrane. The flux of Na+ from lumen
to cell is downhill and active across the peritubular plasma
membrane. When K+ is absorbed, an active K+ uptake step
is necessarily at the apical cell membrane. Protons would be
actively secreted from cells into the lumen. Finally, the steadystate cellular activities of Na+ and K+ were compatible with
a Na+ /K+ pump in the peritubular plasma membrane. In a
study of isolated microperfused late distal tubule of Amphiuma, Stanton et al. (1753) confirmed active acid secretion
toward the luminal fluid, which occurred by an electroneutral amiloride-sensitive Na+ /H+ exchange mechanism in the
luminal plasma membrane of a socalled type I cell. Another
cell type denoted the type II cell, absorbed NaCl in an electroneutral fashion, probably mediated by the parallel operation Na+ /H+ and Cl− /HCO3 − exchangers, that was inhibited
by the thiazide diuretics targeting the anion exchanger (1754,
1755). The type I and type II cells, respectively, were identified by a difference of the peritubular membrane-potential
response to inhibitors added to the luminal perfusion solution.
Neither amiloride nor thiazide affected the electrophysiological properties of the acid-secreting type I cell. In contrast,
these diuretics as well as DIDS hyperpolarized the peritubular plasma membrane of type II cells. Both cell types were
characterized by a fractional resistance of the luminal plasma
membrane near unity, compatible with the ion fluxes through
the luminal plasma membrane being electrically silent (1753).
VT of the above microperfused late distal tubule of Amphiuma
was not significantly different from zero. As previously mentioned, VT increases numerically toward the collecting tubule
(1818), indicating that the studies of Amphiuma and the above
studies of Necturus, respectively, were performed on different
portions of late distal tubule. According to the literature, there
is a remarkable difference between Amphiuma and Necturus.
Cable analysis of the Amphiuma tubule in vitro indicated a
transcellular resistance (RT ) of no more than 72 ·cm2 (1753),
which is more than an order of magnitude lower than that of
the Necturus tubule studied in vivo (26, 1469). Because the
fractional resistance of the luminal membrane of both type I
and type II cells is close to unity (1753, 1755), the low RT
of Amphiuma late distal tubule may have been governed by
paracellular ion fluxes. This, however, deserves further investigations.
Collecting duct system
The collecting tubule is the terminal part of the nephron
that opens into the collecting ducts (Fig. 28). Structurally,
the transition between the late distal tubule and the collecting tubule is gradual, and they have been considered
together as analog segments in non-mammalian vertebrates
(376). In B. bufo, the epithelium of the collecting tubule
and the collecting ducts is heterocellular with principal cells
and intercalated MR cells. The MR cells constitute about
30% of the total cell number of the collecting tubule (1245).
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Osmoregulation and Excretion
There are indications of functions similar to those of MR
cells of the skin. Thus, double immunofluorescence staining
with rabbit anti-Bufo ENaCα antibody and anti-vacuolar-type
H+ -ATPase antiserum showed luminal expression of the Na+
channel at the principal cells and the proton pump at the intercalated mitochondria cells, respectively, of the Japanese black
salamander (1882). Like the principal cells of frog skin, the
uptake of Na+ across the luminal membrane of the principal cells is passive via channels with ENaC-phenotype, that
is, a small-conductance (< 10 pS) amiloride-blockable Na+
channel. The transtubular Na+ uptake is fueled by basolateral
ouabain sensitive Na+ /K+ pumps located in a K+ selective
peritubular membrane. With a lumen-negative VT , the Cl−
flux from lumen to the peritubular space is believed to be passive and to pass the tubular epithelium between the cells (816,
1777, 1778, 1802, 1882). By reabsorbing Na+ and Cl− , the
collecting duct system contributes to the dilution of the urine.
Under certain physiological conditions water is supposed to be
reabsorbed from the diluted tubular fluid, because AVT stimulation results in the translocation of aquaporin AQP-h2K to
the luminal plasma membrane of principal cells (1365) and
the reabsorption of water (575, 1397, 1613).
The collecting duct system has capacity to secrete K+
and H+ via cellular pathways and to participate in the regulation of the body balance of inorganic phosphate (1246,
1247, 1780, 2063). Potassium-loaded Amphiuma tigrinum
kept in 50 mM environmental KCl upregulated (activated
and/or increased the number of) maxi K+ channels and intermediate non-selective cation channels at the luminal membrane supposed to mediate the urinary K+ secretion in hyperkalemic animals (1779, 1781). As previously discussed, with
a plasma pH of about 7.8 and a relatively higher partial CO2
pressure (pCO2 ), the plasma bicarbonate concentration in airbreathing amphibians is larger than in water-breathing animals. Yucha and Stoner demonstrated a dependence of the
lumen-to-peritubular flux of HCO3 − in the initial collecting
tubule on plasma pCO2 , suggesting that tubular acid/base
transporters participate in the regulation of plasma [HCO3 − ].
Because the uptake of HCO3 − was reduced by amiloride or in
Na+ -free perfusion solution, they hypothesized that a luminal
Na+ /H+ exchange mechanism is involved in the transtubular
bicarbonate uptake (2063).
Urinary Bladder
The Wolffian ducts from the kidneys lead the urine directly
into the cloaca, which then runs back into the unpaired thinwalled urinary bladder. The epithelium of the bladder serves
the final adjustment of the ion concentrations of the urine
(4, 1024, 1221, 1262) and participates in the regulation of
whole-body acid-base balance (550) by a carbonic anhydrase
containing minority cell type (1577), presumably MR cells.
In amphibians on land, the bladder constitutes a water storage
organ and during evaporative water loss, water is reabsorbed
from the bladder urine (96, 101, 868, 1757). The significance
of this function is illustrated by the maximal volume of water
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Comprehensive Physiology
in the bladder. For example, expressed in percent of body mass
the bladder capacity of aquatic amphibians like Xenopus,
Triturus cristatus and Necturus maculosus is less than
5% (91, 100), as compared to 50%, or more, in fossorial species like Cyclorana platycephalus, Notaden nicholsi
and Scaphiopus couchi (1144, 1671). Reabsorption of bladder urine is mediated by a distinct urinary bladder aquaporin, AQP-h2 at the luminal plasma membrane of principal
cells (1363).
The luminal surface of the bladder wall is a semi-duallayered heterocellular epithelium including basal cells, principal cells (also denoted granular cells) and intercalated MR
cells. Goblet cells constitute a fourth cell type that contains
mucous filled vesicles (425, 1409, 1643, 1786). The bladder epithelium of Amphiuma contains only three cell types,
with the goblet cells missing. In this species, the MR cells
comprise less than 5% of the total number of cells at the
surface of the epithelium (1262) as compared to 15% in B.
marinus (1643) and 25% in R. catesbeiana (1786). Electron
microprobe analysis of the cellular content of Na+ and K+ ,
respectively, and their response to perturbation of ion composition of the bathing solutions and serosal ouabain exposure
indicated that all four cell types in B. marinus are configured
for vectorial transport with apical Na+ entrance pathways
and basolateral Na+ /K+ pumps (1544). The bladder epithelium is of the high-resistance type with a lumen-negative VT ,
rheogenic active Na+ absorption and active Cl− absorption
under short-circuit conditions (439, 1024, 1262, 1284). The
active Na+ uptake is regulated by aldosterone (348-350). By
the use of a radiolabeled amiloride analogue [3 H]benzamil
and polyclonal anti-Na+ channel antibodies, Kleyman et al.
(946) localized a Na+ channel with ENaC phenotype at the
apical plasma membrane (B. marinus). This was confirmed
by Konno et al. by immunohistochemical localization of the
α-subunit of ENaC. Unexpectedly (conf. 1544), the Na+ channel was expressed at the apical membrane of principal cells
with no staining of MR cells (962).
Aquaporins in Amphibian
Osmoregulatory Epithelia
By governing the hydrosmotic permeability of the above
osmoregulatory epithelia, aquaporins (AQPs) play a fundamental role in the water economy of terrestrial anurans
whether the source of water is environmental water or bladder
urine (1363, 1790, 1792). As indicated above, the cellular sites
of amphibian analogs have been studied in some details. Generally, the transepithelial water permeability is regulated by
insertion of anuran analogues of APQ2 into the apical plasma
membrane of the principal cells. Water leaves the epithelial
cell via the basolateral plasma membrane through constitutively expressed AQP-h3BL (14), which is an aquaglyceroporin that is permeated also by glycerol and urea (7). The
analogs discussed below are specific for anurans and structurally distinct from mammalian APQ2.
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Osmoregulation and Excretion
Aquaporins of ventral skin epithelium
Evaporative water loss
Following AVT stimulation, AQP-2h (B. marinus and
H. arborea) and/or AQP-3h (B. marinus and H. arborea, R.
catesbeiana, R. japonica, R. nigromaculata) are inserted into
the apical plasma membrane of the outermost living cell layer
of water absorbing ventral skin regions (712, 1363, 1366,
1791). Interestingly, AQP2a paralogues resided below the apical plasma membrane in hydrated, non-stimulated anurans,
which might allow for fast transfer to the plasma membrane
when an external water source becomes available (1366).
AQP-h3-like AQPs have been denoted the “ventral skin-type”
AQP because of an exclusive expression in the skin (1363).
In a study of European anurans (R. esculenta, R. temporaria, Bombinator igneus) and urodeles (Triturus taeniatus,
T. cristatus), leading to his Thirty Nine Theses on the Water
Economy of Amphibians and the Osmotic Properties of the
Amphibian Skin, Ernest Overton concluded that water evaporates as from a dilute salt solution depending on temperature,
relative humidity and surface area (1387). This was also the
finding of Edward F. Adolph and Pierre Rey, respectively, who
concluded that the skin does not offer any resistance to water
evaporation into the atmosphere (6, 1529). This notion was
further supported by the observation that live and skinned
frogs lost water by about the same rate (6) and similar to
the rate of water loss by evaporation from replicas made of
agar with the same shape and surface area as live animals.
Although this view was modified subsequently by studying
species of a variety of habitats (1053, 1744, 2045), it has been
accepted generally that the evaporative water loss in amphibians is caused by an unavoidable diffusion of water through
the (more or less) water-permeable skin. However, the methods and experimental protocols of the above classical studies
on live animals (6, 1387, 1529) do not allow for the distinction
between CSF produced by the glands versus a transcutaneous
diffusion of water as the source of EWL. As discussed above
on cutaneous transport mechanisms, the slightly hypotonic
CSF (1002) implies that the transcutaneous flow of water is
directed from the cornified side to the interstitial space driven
by the shallow osmotic gradient and by solute coupled fluid
transport (Fig. 1B). Therefore, EWL stems from fluid produced by subepidermal glands. In a comparative study of 13
anuran species from diverse habitats, Lillywhite and Licht
(1054) observed that curarized anurans exposed to increasing temperatures from 18◦ C to 34◦ C discharged a clear nonviscous fluid that formed a liquid surface film. With some
variation, discharges were more frequent at higher body temperatures. These observations applied to frogs (Hyla cadaverina, Litoria aurea, Rana boylei, Afrixalus brachycnemis), but
not to Bufo woodhousei; all five species mentioned exhibit in
nature basking behavior in sunlight. Nor did curarized nocturnal (H. punctatus, Phyllomedusa tarsius), fossorial (Kaolula
pulchra, Scaphiopus bombifrons, Ensatina eschscholtzi) and
aquatic species (Pipa pipa, Ooedizyga lima) respond by the
above type of gland secretion to increased temperature in
the laboratory (1054). The individual discharge event is the
result of emptying the acinus of a single submucosal gland by
contraction of the thin layer of myoepithelial cells (143). The
sustained flow of ions and water through the acinus epithelium
cannot be recorded by the above method.
As discussed above, the cutaneous surface layer seems
to constitute a regulated external physiological compartment
from which evaporation of water takes place. Thus, the terrestrial environment imposes a demand for a relatively fast
water turnover with potential danger of excessive and irreversible water loss. Owing to the larger surface-area to bodysize ratio, generally obeying the isometric exponent of about
Aquaporins of distal renal epithelia
and urinary bladder
Two AQP’s seem specifically associated with distal epithelia of the anuran kidney: AQP-h2K in H. japonica (1365),
and HC-2 AQP in H. chrysoscelis (2076), respectively. Following AVT stimulation AQP-h2K was translocated to the
apical plasma membrane of the principal cells of the collecting duct, indicating that it governs renal water reabsorption.
Immunofluorescence staining for AQP-h3BL indicated that
the protein is constitutively expressed at the basolateral membrane of the collecting duct principal cells (1365). Besides
the pelvic skin, Tanaka’s group detected AQP-h2 also in
the urinary bladder, which they referred to as the “urinary
bladder-type” amphibian AQP (1363) to distinguish it from
the other isoform that was not found in the urinary bladder of
H. japonica (1806).
Aquaporins of the aquatic Xenopus
The Xenopus AQP-x3 is a homologue of the pelvic skintype AQPa2. However, its gene expression is attenuated at
an identified post-transcriptional step (1363). Therefore, the
gene is not expressed at the protein level, which explains the
low water permeability of the skin of X. laevis (293, 2059).
Another distinctive feature of the water economy of X. laevis
is the vanishing urinary bladder volume capacity (91).
Amphibian Osmoregulation: Adaptations to
Terrestrial Environments
Studies of amphibian water economy have been carried out
for more than 200 years (860). The first study by Robert
Townson indicated that frogs without access to a pool of
free water maintain water balance under terrestrial conditions
(865, 1849). The more recent study by Adolph (6) providing
new quantitative information on amphibian water metabolism
concluded: “It is evident that with respect to water balance
a frog is unsuited to non-aquatic existence.” Studies following Adolph’s and discussed below unanimously showed that,
contrary to his conclusion, amphibians are highly and successfully adapted to a terrestrial life.
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Comprehensive Physiology
0.67 (860), this danger is even more severe for small animals. Numerous studies of amphibian water economy in various terrestrial habitats have disclosed a diversity of mechanisms evolved that would prevent this fatal situation to occur.
The monograph by Hillman et al. (759) contains an extensive review of the large literature on the issue. A brief summary of major principles illustrated by examples is given
below.
Behavioral reduction of body water loss
In the field, EWL rates are strongly influenced by relative
humidity, air temperature, wind speed and by absorbed radiation, but normally not by substrate temperature (1850). As
an exception, the euryhaline leptodactylid frog Thoropa miliaris living in rocky shores maintains its body temperature
3-4◦ C above air temperature by the rocky surface warmed
by the sun (2). Among the above environmental parameters,
temperature and water are probably those that most directly
affect the behavior of terrestrial amphibians. Several species
are nocturnal and by diurnal activity patterns, EWL is reduced
by the animal seeking microhabitats of low temperature, high
relative humidity and low wind speed (412, 780, 1010, 1197,
1478, 1670, 1767). Anurans living in arid habitats escape
the dehydrating environment by burrowing and returning to
the surface during rainfall (255, 256, 435, 780, 896, 1193,
1195, 1587, 1652-1654). Water-conserving posture constitutes another behavior serving a reduced EWL. To reduce
the exposed surface area, the body and chin may be pressed
to the substrate and the limbs tucked under the body (759,
1478, 1829). Other examples include tight coiling of the body
and tail in elongated salamanders (1513) and aggregation
of individuals into groups for reducing the “surface-area to
body-mass” ratio with a tendency to sit on top of each other
with increasing numbers in the group. The crowding behavior
results in a lower EWL than the individual animal otherwise
would experience (19, 869, 1528, 1932).
Waterproofing barriers
The skin resistance to evaporative water loss, rEWL , was introduced for quantifying and comparing EWL in the laboratory
of animals from different terrestrial habitats (83). It has the
dimension of s·cm−1 and is calculated as the reciprocal of
EWL per cm2 of cutaneous surface area per unit of vapor
pressure difference between the animal and the surrounding
air at a specified skin temperature,
rEWL =
ρ
EW L
If EWL is in g·cm−2 ·s−1 , ρ is the vapor-density difference between the animal and the surrounding air in g·cm−3 .
Because of a small solute concentration, the body fluid is
assumed saturated with water vapor at the given temperature.
According to Spotila and Berman (1744), rEWL is partitioned
480
into the internal resistance, ri , and the external boundary-layer
resistance, rb , i.e., rEWL = ri + rb . EWL increases with the
activity of the animal (724) and rEWL is therefore measured
in immobilized animals (mechanically, curarized or pithed)
with emptied urinary bladder, blotted dry with a paper towel,
and placed in a test section of a wind tunnel of relatively
fast laminar air flow (100-200 cm·s−1 ). For estimating the
boundary resistance, replicas of the animals are made in 3%
agar with ri = 0, so that rEWL = rb (1744, 2045). In a recent
study, Lillywhite collected and discussed a large number of
skin resistances to EWL of amphibians, birds and mammals
(1053). The resistance to evaporation of a free water surface is zero. In moist skinned anurans adapted to mesic habitats (Bufo, Rana, Scaphiopus), ri is close to zero and lower
than ri of arboreal frogs, with values ranging from 1.4 to
2.7 s·cm−1 (2044, 2045). A comparative study of 25 species of
Northern Australian frogs belonging to four different families
reported skin resistances to EWL covering the range between
0.6 s·cm−1 and 63.1 s·cm−1 . The frogs were from aquatic,
terrestrial and arboreal habitats, and there was a clear correlation between ecological habitat and rEWL (1851, 2061).
The authors suggested that terrestrial and arboreal species
displaying higher resistances to water loss take advantage of
high environmental temperatures where growth and locomotory speed is higher, without the risk of desiccation. Some
frogs are classified as “waterproof” because their skin resistance to EWL of 300-500 s·cm−1 compares to reptiles with
thick non-hydrated stratum corneum (235, 450, 592, 1053,
1100, 1668, 2015, 2016, 2045). The mechanism seems to be
different for the different species (759). In the South American
tree frog Phyllomedusa, the glands secrete a waxy material of
predominantly wax esters, free fatty acids and hydrocarbons
(145, 623), which is spread over the body surface (1199).
Several studies aimed at the mechanism of intermediate skin
resistances to EWL, but with no clear conclusion (1053).
During periods of aestivation in drying soils, some anurans and urodeles become encapsulated in a cocoon formed
by moultings without casting off the sloughs. Lipids and
proteinaceous materials are deposited between the multiple
superimposed cornified layers (255, 297, 1198, 1586, 2014).
The resistance to water diffusion increases with the number of
layers added. In the South American frog from subtropical and
tropical dry forests, Lepidobatrachus llanensis, ri increased
from ∼0 s·cm−1 in the uncocooned frog to 116 s cm−1 in
an 38-layered cocoon, which was reached after 35 days of
aestivation (1196). Thus, the shedding intervals were about
1.6 day as compared to about 5 days in other anurans at temperatures at about 20◦ C (1011). In Cyclorana australis, the
cocoon impeded water loss to the soil of a significantly lower
water potential than the animal (1531). During a five-month
aestivation period, the lymph concentration of Na+ was kept
almost constant at about 100 mM, while the urea concentration increased from 12 mM to 85 mM. The urea concentration
of bladder urine increased as well, but not as much as that of
the body fluids, thus maintaining the osmotic gradient for
absorption of water from the bladder (1532).
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Comprehensive Physiology
Some amphibians have developed distinctive surface
sculpturing like cutaneous grooves, channels, warts and
prominent elevations denoted verrucae with so-called tubercles and conical short spines (402, 488, 1840). These macroscopic liquid-air interfaces are sites of vaporization and condensation. It is not known how they affect the boundary layer
resistance of evaporation. The question may be a point of
departure in future studies.
Water uptake by cutaneous drinking
Amphibians do not drink by the mouth, but take up water
through the skin. This occurs from a free water surface (102,
105, 865, 1849) or from soil water if the water potential
is higher than that of the body fluids (723, 762, 780-782,
1194, 1530, 1743, 1923). In species of Bufonidae, epidermal
sculpturing increases the rate of rehydration because capillary
forces drive water from the hydration source over the body
surface, which increases the area engaged in water uptake
(293, 1055). The ventral pelvic skin of semiterrestrial anurans denoted the “seat patch” is hypervascularized and is
most important for cutaneous drinking (66, 294, 295, 369,
1195). Comparative studies showed that the pelvic region of
B. bufo, B. alvarius, R. esculenta, R. arvalis, and R. temporaria is more vascularised than the pelvic region of the aquatic
X. laevis (293, 1579). The rate of rehydration increases with
the density of vascularization (293) and arterial perfusion with
Ringer’s solution of the pelvic skin stimulated by AVT in vitro
enhanced the osmotic water influx from artificial tapwater by
maintaining a large osmotic pressure gradient across the epidermis (294). Blood flow through the pelvic region increased
in vivo when dehydrated B. bufo and B. woodhouseii were
placed on a wet substrate (1911, 1912), but not on a dry
substrate (1911). Thus, cutaneous drinking is associated with
enhanced osmotic permeability of the epidermal cells and an
increased blood flow through the capillaries of the seat patch
region. Illustrated above by the hydration status of the animal
and an appropriate hydration source, respectively, cutaneous
drinking is controlled by intrinsic as well as extrinsic factors
(767).
Intrinsic factors Dehydration constitutes an intrinsic
stimulus for cutaneous drinking, and AVT is widely accepted
as a messenger. This is because injection of neurohypophyseal extracts or AVT results in positive body water balance and
enhanced hydrosmotic permeability of the skin and because
the plasma concentration of AVT is increased in dehydrated
animals (21, 22, 92, 93, 100, 209, 212, 515, 727, 728, 861, 961,
1327, 1328, 1839). The neurohypophyseal peptides, hydrins
1 and −2 also stimulate the water permeability of the skin,
to a level similar to that of AVT stimulation (5, 1364). However, dehydration of B. bufo and R. temporaria increased the
rate of rehydration much more than did supramaximal doses
of neurohypophyseal extracts or AVT (862, 872). Furthermore, surgical elimination of the neurosecretory system was
without effect on the water metabolism of B. bufo, which
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Osmoregulation and Excretion
still displayed enhanced water uptake in response to dehydration (873, 874). This would imply that neither AVT nor the
hydrins necessarily play decisive physiological roles in regulating water balance and osmotic permeability of the skin.
The water permeability of the skin monitored directly by the
water flow in vitro or by the rate of rehydration in dehydrated
animals is also under control by the adrenergic nervous system (761, 1499, 1514, 1738, 2057). Hoff and Hillyard (779)
have shown that angiotensin II increases the rate of water
uptake in fully hydrated toads (B. punctatus) that could be
prevented by injection of sarasalsin, which is an inhibitor of
the angiotensin type-1 receptor. Thus, several hormones have
capacity to stimulate the cutaneous water permeability. Their
physiological role and interactions with other intrinsic factors in the water economy of amphibians remain to be fully
established. Interestingly, the urinary bladder filling by itself
constitutes a stimulus for cutaneous drinking. Thus, emptying
the bladder of toads in water balance (B. woodhouseii, B. bufo)
initiated drinking behavior and cutaneous water uptake (868,
1855).
Extrinsic factors and drinking behavior Toads detect
the quality of a hydration source with receptors on their feet
(1767, 1768). Initially, the toad keeps the ventral skin raised
above the substrate so only the feet are in contact with the
substrate. According to Hillyard, the “seat patch up” (SPU)
posture may last for periods of seconds or minutes before
the toad allows the ventral skin to contact the substrate. In
the “seat patch down” (SPD) posture, the ionic or osmotic
properties seem to be evaluated. If the substrate is accepted,
the water absorption response (WR) is initiated where the
hind limbs are abducted and the ventral skin is pressed to the
hydration surface. Dehydrated B. punctatus avoided a hydration source made hyperosmotic by urea (195) or by NaCl
(778), suggesting that receptors in the skin detect the osmolality of the substrate. Hillyard and coworkers analyzed the
sensory response in studies that integrated behavioral observations with membrane potential recordings from principal
cells of the skin, and measurements of the electrical activity
of efferent branches of neural nerves innervating the ventral
skin. The duration of WR was largely unaffected by increasing the NaCl concentration of the hydration source as long as
it was hypoosmotic or not too hyperosmotic to the body fluids. Superfusing the isolated skin epithelium with a 250 mM
NaCl-solution (∼500 mOsm), which suppresses WR in vivo,
depolarized the basolateral membrane and induced trains of
neuronal activity of spinal nerve V and VI. The electrical
responses vanished if 10-μM amiloride was added to the perfusion solution, providing evidence for a Na+ influx through
ENaC as the early event in the sensing of the quality of the
hydration source (763, 766, 1269). In a concomitant paper
(1789), they studied the ability of different salts to suppress
WR behavior at equivalent concentrations (250 mM) of different Na+ -salts. Using the hydration time (i.e., the sum of
SPD and WR) as a measure of the tolerance, a linear increase
in tolerance was observed with increasing molecular weight
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Osmoregulation and Excretion
of the anions tested (chloride, acetate, phosphate and gluconate). It was hypothesized, therefore, that the depolarization of the basolateral membrane is caused by an influx of
Na+ via ENaC with the loop current carried by an inward
flow of anions through opened tight junctions. This was compatible with an observed large and reversible increase in the
transepithelial conductance. Their hypothesis conforms to the
early observation that intercellular junctions of tight epithelia reversibly become leaky if the outside is exposed to a
hyperosmotic solution (298, 497, 1893), and proposes for the
first time a physiological role of the “leaky state” of the skin
epithelium.
Dynamic Aspects of Amphibian
Osmoregulation: Coupling of Skin,
Kidney, and Bladder Functions
Amphibian osmoregulation involves mechanisms primarily
aimed at the volume of the extracellular fluid, and others primarily aimed at the osmolality. They are integrated, although
temporarily one is given priority above the other (29, 489,
863, 864, 1225, 1769, 2065). Extracellular water volume is
governed by the influx through the skin and the bladder wall
balanced by urine production and the voiding of urine stored
in the bladder and, on land, by evaporative water loss (4, 23,
64, 101, 728, 1221, 1489, 1615, 1764, 1765). The studies
discussed below indicate that amphibian water and salt balance, besides being submitted to moderate fluctuations, is
under dynamic, temporal control governed by, for example,
food intake, temperature and environmental osmotic conditions that include transitions between the aquatic and the terrestrial environment and habitats of restricted availability of
free water.
Moderate fluctuations and anticipatory drinking
Table 9 indirectly indicates that the balance of cutaneous
water uptake and water excretion is submitted to variations.
In a direct study, Jørgensen (868) observed variations in
hydration of ±5% of the body mass with empty urinary
bladder in undisturbed B. bufo kept in a simulated terrestrial habitat at 16◦ C. The toads spent most of the time outside the pool of water, where they experienced evaporative
water loss of ∼3% of the body mass per day. Interestingly, the toads initiated cutaneous drinking in a hydrated
state, that is, before EWL dehydrated the body, and generally before the urinary bladder was empty, for which
the phrase anticipatory drinking was introduced, contrasting
emergency drinking in dehydrated animals. The results of this
and a detailed review of other studies led Jørgensen to conclude that normally undisturbed amphibians on land are well
hydrated, underscoring that cutaneous drinking in terrestrial
anurans is not necessarily elicited by dehydration (860, 865,
868, 869).
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Comprehensive Physiology
Food intake
Food consumption initiates drinking, and the water taken up
exceeds the mass of the food eaten, which secures secretion
of fluids with digestive enzymes into the gastrointestinal tract.
In B. bufo, the cutaneous water uptake is proportional to the
size of the meal and may amount to as much as 15% of the
standard body mass. The spadefoot toad (Scaphiopus), which
is active above ground a few days in a year, may consume as
much as 55% of its body mass in a single feeding, which initiates cutaneous drinking in a similar way (435). Eventually,
excess water is stored in the bladder, which allows the animal
to remain hydrated between rainfall periods in the burrows
(1587).
Aquatic versus terrestrial living
Water-acclimated amphibians exhibit high rates of glomerular filtration ensuring the cutaneous uptake of water being
balanced by voiding of highly diluted urine. It has been estimated that the recirculation of bladder urine amounts to less
than 3% of the cutaneous influx of water in water-acclimated
toads (860). During contact with wet soil, the water uptake
by the skin of the terrestrial R. temporaria closely balanced
urine production, so that the reabsorption of bladder urine
amounted to less than 6% of the cutaneous water uptake
(1698). Thus, when environmental water is available, water
permeability of the urinary bladder is small so that reabsorption of water from the bladder urine is of marginal importance
for body water balance. In dry environments, the urine production decreases significantly because of a decreased glomerular
filtration rate and increased fractional water reabsorption both
in anurans (1189, 1624) and in caecilians (1765). In a comprehensive study, Jørgensen concluded that undisturbed toads
only void urine exceptionally, so that the emptying of the urinary bladder “is practically exclusively by resorption through
the bladder wall” (869). Thus, on land, the urine produced
is stored in the bladder and recycled into the body fluids
for maintaining water balance during evaporation of water
into the atmosphere. This is accomplished by an increased
hydrosmotic permeability of the bladder epithelium, which
overcomes the higher osmolality of the bladder urine
(e.g., [1765]).
A two-three-fold stimulation of the cutaneous water permeability of hydrated B. bufo transferred from wet to dry
environment takes place within an hour. Immediately, the rate
of urine production starts to decline. Both of these responses
occur before body water volume decreases. It follows that they
are elicited by exposure to dehydrating conditions per se, not
in response to dehydration. The time course of the water permeability decrease following transfer of hydrated toads from
dry environment to water takes two to five days. However,
the urine production rose immediately after the toads were
transferred to water. Taken together, the above observations
indicate that functions of kidney, bladder and skin in maintaining water balance are regulated in non-dehydrated animals
Volume 4, April 2014
Comprehensive Physiology
(860, 869). The mechanisms that integrate rate of urine production and water permeabilities of skin and bladder wall of
non-dehydrated animals are not known in detail.
Temperature and hibernation
Extracellular volume and osmolality depend on environmental temperature (617, 864, 1116, 1225, 1624). R. catesbeiana
transferred to water at 2-5◦ C initially stopped urine production but not cutaneous water uptake, resulting in a body
mass increase of 5-6%. After about a day, urine production commenced but at a 75% lower rate. This was due to
a significantly decreased GFR from about 34 ml·kg−1 ·hr−1
to 4.5 ml·kg−1 ·hr−1 associated with low tubular reabsorption of water (1624). In the cold temperate zone, amphibians spend about half of the year hibernating, during which
total body water is reversibly increased (166, 870, 1308). A
study on B. bufo kept in 1/25 Ringer’s solution showed that
water uptake at 4◦ C was followed by a delayed cutaneous ion
uptake. At the new steady state, the accumulated fluid volume
amounted to 80% of the standard body mass that was partitioned both to the extracellular (70%) and intracellular space
(10%). Lymph concentrations of Na+ and Cl− were elevated
to levels above their values prior to hibernation, whereas K+
dropped to 1.9 mM and remained low until the temperature
was raised to 20◦ C. Thus, extracellular ion concentrations, as
well, depend on environmental temperature with opposite displacements of the two alkali metal ions (870, 1308). Studies
of overwintering frogs and tadpoles of the aquatic R. muscosa indicated similar but quantitatively smaller water and
electrolyte accumulations (166). The above-mentioned acute
water retention in anurans on cooling to a low temperature
resembles that of dehydrated animals at room temperature.
Isotonic volume expansion of the extracellular fluid prior to
cooling did not prevent the water retention response, which
therefore was suggested to be osmoregulatory rather than volume regulatory. The increased rate of cutaneous ion uptake
seen after a few days at low temperature indicates a reaction
similar to when salt depleted (863, 867).
Body water balance in habitats of restricted
availability of free water
The anurans Scaphiopus spp., B. viridis, X. laevis and R. cancrivora and the urodeles Ambystoma tigrinum and Batrachoseps spp. can cope with osmolalities of the body fluids
that are two to four times above the range of values of other
amphibians (see Table 7), which is associated with reduced
urine flow and storing of hypoosmotic bladder urine. This
enables cutaneous water uptake from surroundings of poor
availability of free water and recycling of water from the
bladder urine into extracellular water. Plasma concentrations
of Na+ , Cl− and especially urea, but not K+ , are elevated
(69, 418, 630, 782, 854, 1191-1194, 1574, 1671) by renal
regulation of volume and composition of the ureteral urine
(1632, 1678). Increased intracellular concentrations of Cl− ,
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Osmoregulation and Excretion
K+ , urea and free amino acids contribute importantly to preventing fatal loss of cell water volume at osmotic equilibrium between intracellular and extracellular fluids (631, 781,
899, 2039). The tolerance to high plasma osmolalities by the
above physiological acclimatizations has expanded the niche
exploitations of amphibians to include deserts and aquatic
environments of high salinity (67, 759, 866, 893).
Reptilia
Reptiles exist and flourish in environments ranging from completely aquatic, both freshwater and marine, to arid terrestrial.
However, there is no common water and electrolyte milieu
intérieur that applies to all orders or species, and the milieu
intérieur in any given species is not as tightly controlled as it
is in birds and mammals (175, 392). Indeed, many systematists do not believe that the reptiles really exist as a separate
class, only as amniotes that are neither birds nor mammals
(1248). However, I continue to treat reptiles as a single class
of vertebrates, like the amphibians, birds and mammals, as
I review their osmoregulation and excretion. The primary
sites involved in osmoregulation are skin, kidneys (including
postrenal modification of the urine in the colon, cloaca, and
bladder) and salt glands.
Function and Regulation of the Physiological
Mechanisms for Osmoregulation
Skin
Skin forms an important potential route for the exchange of
water and, in aquatic reptiles, ions with the external environment. The appearance of reptilian skin suggests that it
might be impermeable to water, as it was once thought to be.
But it is not. Indeed, as shown in Table 11, it is the principal route for evaporative water loss (∼70-90% of the total)
in semiaquatic and terrestrial reptiles (103, 280). Even in
aquatic reptiles, the integument is highly permeable to water
(462, 464, 1566). The permeability of the skin to water, determined as the rate of evaporative water loss under identical
conditions, decreases with increasing aridity of the habitat
(also often reported as increasing resistance to water loss
with increasing aridity), a pattern observed across orders and
across species in a single order (Tables 1 and 2) (103, 437,
579, 1053, 1186, 1187, 1559, 1560) (see also Table 2 in Lillywhite [1053] for a more exhaustive list). This relationship
has also been observed within geographically isolated populations of a single species (438). Moreover, for some species the
rate of evaporative water loss across the skin has been shown
to decrease when they are exposed to an arid environment
(891, 952).
The permeability of the integument of aquatic species to
water decreases as the salinity of their normal habitat increases
and this holds across orders and across species (Table 12)
(462, 464). However, a puzzling observation at odds with all
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Table 11
Comprehensive Physiology
Examples of Cutaneous Evaporative Water Loss in Living Reptiles in Dry Air
Cutaneous Water Loss
Normal Habitat
mg cm−2 day−1
Percent TEWL
Semiaquatic, freshwater
32.9±2.45 (8)
87±2.1 (8)
Pseudemys scripta
Semiaquatic, freshwater
12.2±1.44 (6)
78±2.7 (8)
Terrapene Carolina
Terrestrial, mesic
5.3±0.41 (6)
76±3.4 (6)
Iguana iguana
Terrestrial, mesic
4.8±0.50 (8)
72±4.3 (8)
Saurmalus obesus
Terrestrial xeric
1.3±0.10 (6)
66±2.0 (6)
Species
Crocodilia
Caiman sclerops
Testudinea
Squamata
Sauria
Values are means ± SE; numbers in parentheses equal number of animals; TEWL equals total evaporative water loss. Data are from Bentley and
Schmidt-Nielsen (103). Table from Dantzler and Bradshaw (392) with permission.
other observations is that two species of sea snakes (Hydrophis
ornatus and H. inornatus) have an integument nearly as permeable to water as that of purely freshwater snakes (Table 12).
Because these two species maintain the osmotic and ionic
composition of their plasma far away from that of sea water
Table 12
and similar to that of other sea snakes, the physiological significance of this observation is unclear (462). The skin of aquatic
reptiles also has a substantial permeability to sodium, and, at
least among snakes, it is significantly greater in freshwater
species than in estuarine or marine species (462). The high
Examples of Efflux and/or Influx of Water in Reptiles in Seawater
Normal Habitat
Water Efflux
ml 100g−1 h−1
Water Influx
ml 100g−1 h−1
Semiaquatic, freshwater
—
0.72±0.11 (3)
Semiaquatic estuarine
0.16±0.05 (11)
0.17±0.03 (11)
Nerodia sipedon
Semiaquatic, freshwater
1.54±0.54 (5)
1.33±1.19 (5)
Nerodia fasciata
Pictiventris
Semiaquatic, freshwater
2.84±2.00 (4)
2.54±2.49 (4)
Acrochordus
Granulatus
Semiaquatic, estuarine
0.49±0.04 (4)
0.47±0.19 (4)
Laticauda
Laticauda
Aquatic, seawater
0.20±0.06 (4)
0.17±0.05 (4)
Hydrophis belcheri
Aquatic, seawater
0.61±0.06 (2)
0.54±0.21 (2)
Hydrophis ornatus
Aquatic, seawater
1.26±0.27 (8)
1.08±0.32 (8)
Hydrophis
Inornatus
Aquatic, seawater
1.18±0.52 (4)
1.19±0.28 (4)
Species
Testudinea
Chrysemys picta
Malaclemys
Terrapin
Squamata
Ophidia
Values are means ± SD. Numbers in parentheses equal number of measurements. Data were determined from isotopic fluxes. Data on
Testudinea are from Robinson and Dunson (1566); those on Squamata are from Dunson (462). Adapted from Dantzler and Bradshaw (392) with
permission.
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Table 13
Osmoregulation and Excretion
Examples of Water Loss Through Shed Skin of Reptiles
EWL mg cm−2 h−1
Species
Normal Habitat
Untreated
Extracted
Lipid Content % dry weight
Acrochordus
Javanicus
Aquatic, freshwater
0.50±0.08 (10)
1.30±0.13 (8)
2.43
Nerodia
Rhombifera
Semiaquatic, freshwater
0.41±0.13 (7)
2.62±0.50 (9)
4.30
Elaphe
Obsolete
Terrestrial, mesic
0.22±0.01 (39)
2.53±0.34 (44)
5.91
Crotalus
Adamanteus
Terrestrial mesic
0.22±0.07 (17)
2.59±0.33 (21)
5.89
Crotalus
Viridis
Terrestrial semi-arid
0.14±0.04 (4)
2.92±0.38 (6)
7.40
Crotalus
Cerastes
Terrestrial xeric
0.16±0.04 (8)
2.40±0.40 (10)
8.61
Terrestrial, mesic
1.16±0.09 (5)
1.98±0.12 (4)
Ophidia
Sauria
Iguana iguana
Values are means ± SE; numbers in parentheses equal number of measurements; EWL equals evaporative water loss. Data are from
Roberts and Lillywhite (1559, 1560). Table from Dantzler and Bradshaw (392) with permission.
rate of sodium influx that this permits in freshwater species
may explain their intolerance to seawater (462).
Within reptilian skin, the epidermis forms the limiting
barrier for water exchange and, in aquatic species, for ion
exchange with the environment. Within the epidermis, lipids
form the major barrier for the diffusion of water (681, 682,
1559, 1560, 1775). Extraction of lipids from the shed skins of
ophidian and saurian reptiles eliminates the barrier to evaporative water loss (Table 13), but denaturation of proteins has
little effect (1056, 1559, 1560).
The lipids involved in restricting water permeation are
found in multiple bilayer sheets, consisting primarily of
highly saturated, unbranched, long-chain ceramides extruded
by lamellar granules into the extracellular compartment of
the mesos layer of the epidermis (987, 1019, 1053, 1056,
1559, 1967). In reptiles, these epidermal lipids also include
the unusual glycolipids, glucosylsterol and acylglucosylsterol
(3). It is not yet known whether the lipid composition differs between species from arid and mesic environments or
changes with adaptation of a single species to a more arid or
more mesic environment. However, Roberts and Lillywhite
(1560) found that total lipid content of the ophidian epidermis increases as water permeability decreases (Table 13), and
it is possible that the quantity of lipids is more important than
their composition in determining water permeability.
Recently, the water channel aquaporin 3 (AQP3) has been
found in mammalian epidermis (1737), although it appears
to be involved in maintaining epidermal hydration, not in
transepidermal water permeability. Whether an ortholog of
AQP3 or any other water channel that could play a role in
Volume 4, April 2014
water permeability is present in reptilian epidermis has yet to
be determined.
The way in which inorganic ions permeate the skin of
aquatic reptiles is not at all clear. In general, movement of
inorganic ions across lipid membranes requires protein channels or transporters, and no such channels or transporters have
been described in reptilian epidermis. Extraction of lipids
from shed ophidian skin markedly increases the permeability to inorganic ions (1775), as would be expected, but this
does not explain how inorganic ions cross the skin when the
lipids are present, especially in view of the observation that
extraction of proteins alone has little or no effect on the permeability of inorganic ions (1775). In addition, Stokes and
Dunson (1775) reported that there was directional asymmetry in tracer fluxes of sodium (as well as water) across shed
skins that were abolished by lipid extraction. This finding is
controversial and has yet to be confirmed, but it suggests that
some aspect of lipid composition could actually play a role in
ion permeability across the epidermis.
No information is available about any physiological regulation of ion and water movement across reptilian integument,
although it is possible that regulation of vascular perfusion of
the skin could influence such movements. Any effect would,
of course, depend on the relationship of the vascular rate
of delivery of ions and water and the permeability of the
epidermis to them. No information on the regulation of
vascular perfusion or its possible effects on magnitude of
ion and water movements across the epidermis is available
for reptiles. It should also be noted that almost all information available about ion and water exchange across the
485
Osmoregulation and Excretion
reptilian skin has come from studies on squamates, primarily ophidians; very little is from studies on chelonians and
crocodilians.
Kidneys
The kidneys play a critical role in regulating the composition
of the internal environment of reptiles, as they do in other
tetrapod vertebrates, by controlling the excretion of water,
ions, and nitrogenous wastes. However, in all reptile species,
structures distal to the kidneys (colon, cloaca or bladder) can
modify the composition of the urine before it is excreted,
and, in some reptile species, salt glands contribute to the
excretion of ions. Nevertheless, the kidneys are quantitatively
more important than any other structures in regulating the
output of ions and water and, therefore, the composition of the
internal environment. This regulation involves adjustments
both in filtration at the renal glomerulus and in reabsorption
and secretion along the renal tubules.
General form of kidneys and nephrons
The external shape of reptilian kidneys varies extensively,
apparently being determined by the marked variation in body
type between the different orders of the Class Reptilia. For
example, the kidneys of snakes are long and thin, those
of lizards are compact, somewhat triangular-shaped organs
joined at the posterior end, and those of turtles are shaped by
the carapace (W. H. Dantzler, personal observations) (177).
However, the basic components of the nephrons, the actual
functional units of the kidneys, are the same in all reptiles. Each includes a glomerulus (except for a few nephrons
observed in one species of lizard [1349] and in some snakes
[1515]); a short, ciliated neck segment; a proximal tubule; a
short, ciliated thin intermediate segment; and a distal tubule
(382). Reptilian nephrons do not have the long loops of Henle
arranged parallel to collecting ducts that are found in avian
and mammalian nephrons. Indeed, the gross, external shape of
the kidneys constrains the gross arrangement of the nephrons
within them. For example, in lizard kidneys the nephrons
branch obliquely off collecting ducts and are arranged in compact bunches (1349), whereas in snake kidneys the nephrons
lie side by side in neat parallel rows and attach to the major
collecting ducts at roughly right angles (382). Despite these
differences in gross nephron arrangement in the kidneys of
the different orders of living reptiles, in all cases examined,
the nephrons are arranged so that the beginning of the distal
tubule is closely apposed to the vascular pole, apparently the
afferent arteriole, of its own glomerulus (S. D. Yokota, R. A.
Wideman, and W. H. Dantzler, unpubl. observ.) (810, 1349).
Finally, it is important to note that in all reptiles studied,
the capillary network surrounding the renal tubules receives
blood not only from the glomerular efferent arteriole but also
from a renal portal system supplying venous blood from the
posterior regions of the body.
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Comprehensive Physiology
Glomerular function
The initial process in the formation of urine is the delivery of
water and solutes from the blood to the lumen of the proximal
renal tubules. In glomerular nephrons (almost all reptilian
nephrons), this involves the ultrafiltration of plasma water
and solutes across the glomerular capillary walls, a process
first documented in reptiles in 1933 by Bordley and Richards
(152). They collected fluid from Bowman’s space around the
glomerular capillaries and found it to be protein-free (within
the limits of their measurements) and to contain small solutes
in the same concentrations as in the plasma. The composition of the filtrate determines the initial composition and the
rate of formation of the filtrate determines the initial volume
flow rate of the tubular fluid. The production of this ultrafiltrate by arterial hydrostatic pressure maintained for other
purposes is an efficient way to excrete large volumes of fluid
without expending additional energy specifically for this purpose. Moreover, because the rate of formation of glomerular
filtrate can be varied readily (see below), it can be an important determinant of the volume of urine excreted. Those few
aglomerular nephrons observed in some reptilian species, if
they function at all, must do so by secreting ions and water
across the tubule epithelium into the lumen (see below), but
these nephrons are clearly not of much significance in the
formation of reptilian urine.
The rate of ultrafiltration at the glomerulus of a single
nephron (SNGFR) is the product of the ultrafiltration coefficient (Kf ) and the net ultrafiltration pressure (PUF):
SNGFR = K f [(PGC − PBS ) − πGC ]
Within the brackets, PGC is the outwardly directed hydrostatic
pressure in the glomerular capillaries, PBS is the inwardly
directed hydrostatic pressure in Bowman’s space, and π GC is
the colloid osmotic pressure in the capillaries, which opposes
filtration. The sum of these pressures is the PUF. Almost no
protein is normally filtered and, therefore, essentially no colloid osmotic pressure exists outside the capillaries to favor
filtration. Yokota, working with anesthetized garter snakes
(Thamnophis spp.), measured (in mm Hg) a mean arterial
pressure of 38, a mean PGC of 22, a mean PBS of 2, and a
mean π GC in the afferent arteriole of 17 (S. D. Yokota, pers.
comm.). These data, the only such measurements available
for reptiles, yield a PUF of 3 mmHg at the afferent end of
the glomerular capillary network. Because Yokota made the
PGC measurements randomly in the glomerular capillaries,
it seems probable that, as in mammals, PGC falls only very
slightly along the length of the glomerular capillary network.
This nearly constant PGC is maintained by resistances in the
afferent and efferent arterioles. As filtration occurs along the
length of the capillary network, π GC rises. Whether or not it
rises sufficiently in garter snakes to equal PGC , thereby causing filtration to cease before the efferent end of the capillary
network, is unknown. However, because the value of PUF
at the afferent end of the glomerular capillaries is so low, it
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Comprehensive Physiology
seems likely that this does occur in garter snakes and possibly in other reptiles. If this filtration equilibrium is normally
reached before the efferent end of the capillary network (i.e.,
not all of the network is used for filtration), the SNGFR will
be particularly sensitive to changes in renal plasma flow.
As indicated above, SNGFR is a function, not only of
PUF, but also of Kf , which itself is a function of the surface
area of the capillaries available for filtration (A) and the aqueous permeability of the capillary wall (hydraulic conductivity
or Lp ). A measurement of either A or Lp would permit determination of the other from that value, the PUF, and the SNGFR.
However, at present, it is not possible to measure either A or
Lp accurately for any species. Therefore, it is only possible
to calculate their product, Kf , from the PUF and the SNGFR.
In the case of garter snakes, the only reptile for which data
are available, a PUF of 3 mmHg and SNGFRs ranging from
0.6 to 5 nl/min (152) (S. D. Yokota and W. H. Dantzler, unpubl.
observ.) yield Kf values of 0.2 to1.7 nl/min/mmHg (392). The
larger value is only about one-third that in Munich-Wistar rats
(196, 411), less than one fourth that in Congo eels (Amphiuma means) (1432), but about equal that in river lampreys
(Lampetra fluviatilis) (1216).
The rate of formation of glomerular filtrate for the whole
kidney (GFR) is the sum of all the SNGFRs for that kidney.
However, it is not possible to determine experimentally all
the SNGFRs and sum them in the kidneys of living animals.
Fortunately, it is possible to determine the whole-kidney GFRs
(for both kidneys combined) by clearance methods in living
animals.
A number of whole-kidney GFR measurements have been
made in some members of all four extant orders of living reptiles during acute changes in hydration or during a water load
(usually hypoosmotic glucose solution) or a salt load (usually
1 mol/l hyperosmotic sodium chloride) (Table 14) (see also
table 8.1 in [189]). In general, whole-kidney GFRs tend to
increase with a water load and decrease with dehydration or
a salt load, but there is considerable variation among species,
particularly in response to a salt load, where an increase in
GFR frequently occurs (Table 14). This latter variation in
response to a salt load may reflect the fact that a hyperosmotic
salt load in a well-hydrated animal, although given to mimic
dehydration, would initially tend to lead to plasma expansion,
increased renal blood flow, and increased GFR. Only with prolonged infusion, which was not generally performed, would a
salt load in a well-hydrated animal lead to water and volume
depletion, an increase in plasma osmolality and a decrease
in GFR. Also, lingual salt glands (see below) in crocodiles
and wholly aquatic sea snakes may have removed sodium
chloride rapidly enough to make the salt load equivalent to
an isosmotic plasma expansion or even a hypoosmotic water
load, thereby leading to an increase in GFR. In some studies
(Table 14), renal function was observed to cease completely
when plasma osmolality increased sufficiently, thereby eliminating renal water excretion at the expense of retaining salts
and metabolic wastes. However, this occurred at much higher
plasma osmolality in the desert tortoise than in semiaquatic
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Osmoregulation and Excretion
or mesic species, suggesting that these desert animals tolerate
much wider swings in plasma osmolality than other species
(395, 1286). In summary, it appears that reptiles, in contrast to mammals and despite some variation among species,
show marked increases or decreases in whole-kidney GFR
with acute physiological changes in hydration, suggesting
that changes in GFR play an important physiological role in
regulating the volume and composition of the final urine (see
below).
These changes in whole-kidney GFR appear to result primarily from changes in the number of filtering nephrons,
although variation in the filtration rate of those nephrons filtering probably also plays some role (381, 395, 1623, 2056).
The concept that a change in the number of filtering nephrons
is the primary process involved in a change in whole-kidney
GFR in reptiles is supported indirectly in those species studied by histological evidence that the ratio of open to collapsed proximal tubule lumina correlates roughly with the
whole-kidney GFR (1623) and by data showing that the maximum rate of tubular transport of a secreted substance (paraaminohippurate, PAH) varies directly with whole-kidney GFR
(381, 395). Of greater significance, direct quantitative measurements of blood flow rates in single glomeruli of garter
snakes (Thamnophis sirtalis) confirm the presence of intermittent blood flow and, presumably, of intermittent filtration,
and also show that the fraction of glomeruli with intermittent blood flow increases with increasing plasma osmolality
(2056). Because plasma osmolality also increases with dehydration, these correlations strongly support the concept that
the decreases in whole-kidney GFR observed with dehydration are the result of decreases in the number of filtering
nephrons. Because the release of arginine vasotocin (AVT),
the antidiuretic hormone in reptiles, is also stimulated by
increases in plasma osmolality (178, 392), this further correlation strongly suggests that AVT plays a role in producing glomerular intermittency during dehydration, at least in
ophidian reptiles. In further support of this concept, intravenous infusions of AVT in garter snakes cause blood flow in
individual glomeruli to cease (Fig. 29) (2056) just as they produce decreases in whole-kidney GFR in water snakes (Natrix
sipedon) (381). Varying the whole-kidney GFR by varying
the number of nephrons filtering seems reasonable in animals
whose nephrons are not structured to function in concert to
produce urine hyperosmotic to plasma. Moreover, as noted
above, reptiles have a renal portal system, which can supply the cells of non-filtering nephrons in the absence of a
postglomerular arterial blood supply.
Mechanistically, decreases in blood flow in individual
glomeruli in garter snakes apparently result from constrictions of the afferent glomerular arteriole in response to AVT
(either endogenous or exogenous) interacting with V1 -type
receptors (Fig. 29) (2056). When the diameter of the afferent arteriole becomes sufficiently small, the rigid, nucleated
red blood cells cannot pass, occluding the vessel and causing cessation of flow and filtration (Fig. 29) (2056). However, filtration at an individual glomerulus may actually cease
487
Osmoregulation and Excretion
Table 14
Comprehensive Physiology
Changes in Whole-Kidney GFR
Condition
GFR ml kg−1 h−1
References
Crocodylus johnsoni
(freshwater, semiaquatic)
Control
Dehydration
Water load
6.0±1.5
1.9±0.2
3.3±1.1
(1623)
Crocodylus porosus
(salt water, semiaquatic)
Control
Salt load
Water load
1.5±0.2
2.8±0.9
18.8±2.3
Control
Salt load
No urine flow when plasma osmolality
↑ 100 mOsm
Water load
4.7±0.60
2.9±0.91
Control
Salt load
No urine flow when plasma
osmolality ↑ 20 mOsm
Water load
4.7±0.69
2.8±0.90
Tiliqua scincoides
Blue-tongued lizard
(terrestrial)
Control
Dehydration
Salt load
Water load
15.9±1.0
0.7
14.5±0.5
24.5±2.0
(1623)
Phrynosoma cornutum
Horned lizard
(arid, terrestrial)
Control
Dehydration
Salt load
Water load
3.5±0.323
2.1±0.20
1.7±0.40
5.5±0.54
(1562)
Hemidactylus sp.
Puerto Rican gecko
(moist, terrestrial)
Control
Dehydration
Salt load
Water load
10.4±0.77
3.3±0.37
11.0±2.18
24.3±1.67
(1562)
Salt load
No urine flow when plasma osmolality
↑ 50 mOsm
Water load
13.1±1.26
(379, 381)
Control
Salt load
Chronic intraperitoneal seawater load
Water load
Chronic intraperitoneal water load
0.78 (0.49-2.78)
2.24 (1.41-6.42)
7.05±(6.26-7.83)
0.17±(0.03-0.35)
5.67±(4.40-6.20)
(226)
Control
Dehydration
Water load
3.9
3.4
4.8
(1627)
Species and Habitat
Crocodilia
Testudinea
Gopherus agassizii
Desert tortoise
(arid, terrestrial)
Pseudemys scripta
Freshwater turtle
(fresh water, semiaquatic)
(395)
15.1±.64
10.3±2.00
Squamata
Sauria
Ophidia
Nerodia sipedon
Freshwater snake
(fresh water, semiaquatic)
Aipysurus laevis
Olive sea snake
(salt water, aquatic)
22.8±1.75
Rhynchocephalia
Sphenodon punctatum
(moist terrestrial)
Values are means or means ± SE except for sea snakes where, because data did not show a normal distribution, the values are given
as medians and interquartile ranges. All means with SE and medians are for 4 or more values. Modified from Dantzler and Bradshaw (392) with
permission.
488
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Comprehensive Physiology
Osmoregulation and Excretion
Afferent arteriolar diameter
(μ)
20
15
= 32% Initial cross–sectional area
10
= 16% Initial cross–sectional area
5
AVT
Single-nephron blood flow rate
(nl/min)
80
60
40
20
AVT
–2
0
2
4
6
Time (min)
Figure 29
Simultaneous relationship of afferent arteriolar diameter and blood flow for two
representative nephrons in snake (Thamnophis sirtalis) kidney during continuous infusion of arginine vasotocin (AVT). Arrows mark the start of AVT infusion at a rate of 17 pg 100 g−1 min−1 .
Reproduced from Yokota and Dantzler (2056) with permission.
before the blood flow ceases or may not occur at all during
periods of continuous low blood flow. This could occur if
the outwardly directed hydrostatic pressure in the glomerular capillaries decreased sufficiently to equal the sum of the
opposing colloid osmotic pressure of the plasma proteins and
the hydrostatic pressure in Bowman’s space (i.e., PUF fell to
zero). This seems likely in garter snakes given their low PUF
and quite likely in other reptiles as well, although PUF has
not been determined in any other species.
In addition to AVT, the hormone prolactin may play a role
in regulation of glomerular blood flow and, thus, filtration. In
some freshwater turtle species (definitely in Chrysemys picta
and possibly in Pseudemys scripta), prolactin administration
increases whole-kidney GFR in intact animals and reverses
the decrease in whole-kidney GFR observed in hypophysectomized ones, possibly by relaxing the afferent arteriole (197).
It is also possible that there is some neural control of glomerular blood flow in some ophidian reptiles. Inhibitor studies in
sea snakes (Aipysaurus laevis) and garter snakes suggest that
α-adrenergic agonists, whose release is stimulated by high
plasma potassium concentrations, may regulate GFR by constricting the afferent arteriole (S. Benyajati, S. D. Yokota, and
W. H. Dantzler, unpubl. observ.) (108, 2055). However, more
direct studies are required to determine the exact roles, if any,
of both prolactin and α-adrenergic nerves in regulation of
glomerular blood flow and filtration.
Autoregulation, the process in mammals whereby mechanisms intrinsic to the kidney maintain both renal blood flow
Volume 4, April 2014
and GFR relatively stable independent of changes in mean
arterial blood pressure, has yet to be examined in reptiles.
However, it seems unlikely to be significant in view of the
highly variable mean arterial pressure, variable SNGFRs,
intermittent nephron function and lack of a macula densa (382,
1316, 1726) (despite apposition of the early distal tubule to
the vascular pole of its own glomerulus). However, the study
of Yokota and Dantzler (2056) suggests individual glomeruli
in snake kidneys may always have either high or low blood
flow and, thus, high or low SNGFR, and this may be regulated
in some way.
Tubule function
The renal tubules modify the initial glomerular filtrate by
the processes of reabsorption and secretion, thereby determining the final volume, ionic composition and osmolality
of the ureteral urine. The following discussion is limited to
tubule transport of sodium, potassium, water and the primary
excretory end products of nitrogen metabolism.
Inorganic ion transport Sodium. Although neither the
concentration of sodium in the extracellular fluid nor the extracellular fluid volume itself is maintained as constant in reptiles
as in mammals (173), maintaining total body sodium within
some broad general range, primarily by regulating excretion,
is still critically important for normal physiological function.
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Osmoregulation and Excretion
Most of this regulation occurs via renal tubule transport of
sodium.
Although clearance studies based on collections of
ureteral urine show that reptilian renal tubules may reabsorb
50% to 98% of the filtered sodium, depending on the species
and the physiological circumstances of the experiments, on
average at least 10% of the filtered sodium escapes reabsorption (382, 392, 1776). This is far more than in mammals
where less than 1% normally escapes reabsorption. However,
further regulation of sodium excretion in reptiles can occur in
the bladder, cloaca or colon (see below). Moreover, the clearance studies in reptiles do not include any filtered sodium that
is contained in urate precipitates in uricotelic reptiles (see
below) and, therefore, may actually overestimate the fraction
of filtered sodium reabsorbed by the renal tubules. In addition, clearance studies only indicate net sodium transport, not
whether both secretion and reabsorption of sodium occur.
In reptiles, in contrast to mammals, the proximal tubule
is not necessarily the site where the largest fraction of filtered sodium is reabsorbed. In vitro microperfusion studies of
snake (Thamnophis spp.) renal tubules and in vivo micropuncture studies of lizard (Sceloporus cyanogenys) renal tubules
suggest that only about 35-45% of filtered sodium is reabsorbed along the proximal tubules whereas about 50-70% is
reabsorbed along the distal portions of the nephrons (382, 390,
1776). The fraction reabsorbed in a given segment reflects the
relationship among the amount of filtered sodium reaching
that segment, the length of the segment and rate of reabsorption per unit length. The few data available suggest that
the rate of sodium reabsorption is less than or equal to that
of mammals along the proximal tubule and equal to that of
mammals along the distal tubules (382, 390, 1776).
Essentially no direct information is available about the
mechanism of sodium reabsorption in reptilian proximal
tubules, and the steps in the transport process must be inferred
from a few measurements on garter snake (Thamnophis spp.)
tubules and analogy to other vertebrates. A very small, lumennegative transepithelial potential (ca. −0.50 mV) in proximal tubules indicates that transepithelial sodium transport is
against an electrochemical gradient (388). Moreover, because
fractional reabsorption of chloride is the same as that of
sodium in the proximal tubule (390), it appears that transepithelial transport of sodium chloride involves active transport
of sodium and passive transport of chloride. This may be true
for other reptile species as well.
A basolateral membrane potential of about −60 mV (924)
in garter snake proximal tubules indicates that, as in other vertebrates, sodium can enter the cells from the lumen down an
electrochemical gradient. Also, because a functional sodiumhydrogen exchanger has been demonstrated in brush border
membrane vesicles from these kidneys (396), it seems very
likely that, as in mammals, luminal sodium entry primarily
involves such exchange. This possibility is indirectly supported by observations that administration of a carbonic anhydrase inhibitor to water snakes (Nerodia sipedon) results in
ureteral urine that is alkaline and contains increased amounts
490
Comprehensive Physiology
of sodium and potassium (379). In addition to entering the
cells across the luminal membrane by exchange for hydrogen, some sodium almost certainly enters coupled to the entry
of glucose, amino acids, phosphate and other molecules. As
in other vertebrates studied, transport of sodium out of the
cells against an electrochemical gradient at the basolateral
membrane almost certainly involves Na+ -K+ -ATPase, which
is present in the basolateral membrane of proximal tubules in
snakes (107, 380) and most likely other reptiles.
In vitro microperfusion studies of snake (Thamnophis)
distal tubules (117, 119, 122) provide insights into a possible
intrinsic control mechanism for regulating sodium reabsorption in this tubule segment. These studies reveal a substantial
lumen-negative transepithelial potential (ca. −35 mV), which
is inhibited by ouabain on the basolateral side or amiloride on
the luminal side. The calculated short-circuit current (considered to represent sodium reabsorption) and the transepithelial
potential, while dependent on the presence of sodium in the
lumen, both decay rapidly when the luminal sodium concentration exceeds 30 mM. The decays in transepithelial potential and short-circuit current appear to represent an increase
in resistance to sodium transport through the active transport
pathway. Under these conditions the rate of active sodium
extrusion across the basolateral membrane is not great enough
to keep up with the rate of passive sodium entry across the
luminal membrane, and the cells swell. This intrinsic response
to a large sodium load in the distal tubules may help prevent
the reabsorption of too much sodium at a time when there is a
need to excrete excess sodium. Also, a transport system poised
to work effectively only at low luminal sodium concentrations
will enhance dilution of tubule fluid even when the luminal
sodium concentration is already low and thus enhance the
excretion of excess water (see below). Whether such intrinsic
controls are available in the distal tubules of other reptiles is
unknown.
Hormonal regulation of renal tubule transport of sodium is
far from clear. In clearance studies, administration of physiological doses of reptilian antidiuretic hormone, arginine vasotocin (AVT), increases fractional reabsorption of sodium in
a number of species, but not all (392). In fact, in one chelonian species (Chrysemys picta belli), administration of AVT
actually decreased fractional sodium reabsorption (231). Even
if AVT tends to stimulate sodium reabsorption in most reptile species, the tubule site of such stimulation is completely
unknown.
Although aldosterone and corticosterone are the major
secretory products of the reptilian adrenals (1600, 1913), their
action on renal tubule sodium transport—in fact, on kidney
function in general—is not well understood and appears to
vary among species, even of the same order (392). For example, as in mammals, aldosterone appears to stimulate tubule
sodium reabsorption in the freshwater snake Natrix cyclopion (1026) and the lizard Varanus gouldii (173, 1537), but
it may have the opposite effect in the lizards Ctenophorus
ornatus and Dipsosaurus dorsalis (174, 179, 268). Moreover,
even when aldosterone appears to stimulate tubule sodium
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Comprehensive Physiology
reabsorption, the tubule site of such stimulation is unknown.
Corticosterone also has variable effects on different lizard
species (392). Clearly, understanding hormonal regulation of
sodium transport in reptiles requires much more study.
Potassium. Potassium, the major cation of the intracellular fluids, although particularly critical for the function of
excitable cells, is not maintained as constant in reptiles as in
mammals. However, there is still considerable regulation of
the total quantity in reptiles, largely by renal and extrarenal
excretion. Renal clearance studies in numerous species (392)
and renal micropuncture studies in one species (blue spiny
lizard, Sceloporus cyanogenys) (1776) indicate that either
net tubule secretion or net tubule reabsorption may occur.
Although the magnitude and sites of tubule potassium transport are not well described for most reptiles, the micropuncture study on Sceloporus cyanogenys (1776) suggests that
about 25-35% of filtered potassium may regularly be reabsorbed along the proximal tubule under normal circumstances.
Along the distal tubules, transport may vary from net reabsorption of 20% of the filtered load to net secretion of as
much as 180% of the filtered load, most likely in response to
varying needs to retain or excrete potassium. This pattern in
Sceloporus distal tubules is similar to that observed in mammals and those other non-mammalian vertebrates that have
been studied (382). However, the exact distal tubule sites,
cell types, mechanisms or regulation (hormonal or otherwise)
involved in transepithelial tubule transport of potassium in
Sceloporus are unknown. Some additional potassium can be
reabsorbed in the collecting ducts in Sceloporus, but this does
not appear to be very significant (1776). Finally, as a cautionary note about the clearance and micropuncture studies,
it is important to recognize that in uricotelic reptiles, some
potassium in the tubule fluid may be combined with urate
precipitates (see below) (387), and therefore, measurements
involving only the aqueous phase of the urine may not give
an accurate picture of the magnitude or even the direction of
net tubule transport (387).
Water Transport Water reabsorption. Water reabsorption by the renal tubules plays a critical role in the overall
water balance of reptiles. Sometimes it occurs at the same
rate as solute reabsorption (as isosmotic fluid) and sometimes
it lags behind, depending on the need to conserve or excrete
water. However, in reptiles, total water reabsorption by the
renal tubules, measured as a fraction of the filtered fluid, never
equals, much less exceeds, 99%, even during dehydration, as
it does in mammals (189, 383). Moreover, microperfusion,
micropuncture and clearance studies indicate that only about
30-50% of the filtered fluid is reabsorbed along the proximal tubules in ophidian, saurian, chelonian and crocodilian
reptiles (390, 395, 1629, 1776). This is distinctly below the
minimum of at least two-thirds of the filtered fluid normally
reabsorbed along the proximal tubules of mammals (188).
Instead, perhaps as much as 45% of the filtered fluid can be
reabsorbed along the distal tubules and collecting ducts, the
exact amount often depending on the need to conserve water
Volume 4, April 2014
Osmoregulation and Excretion
and the action of antidiuretic hormone (188, 392). In considering the overall fluid balance of reptiles, however, it must be
remembered that substantial amounts of water can be reabsorbed in the colon, cloaca or bladder, depending on the needs
of the animal (see below).
The mechanism by which filtered fluid is reabsorbed in
the proximal tubules of reptiles is not understood, although
microperfusion studies in snakes (Thamnophis spp.) and
micropuncture studies in lizards (Sceloporus cyanogenys)
indicate that the fluid in the proximal tubule remains isosmotic with the plasma during reabsorption (390, 1776). However, although these studies indicate that sodium and water
can be reabsorbed at osmotically equivalent rates, they do not
indicate that fluid reabsorption always depends on sodium
reabsorption. Indeed, in snake (Thamnophis spp.) proximal
tubules perfused in vitro, isosmotic fluid reabsorption can
occur when lithium replaces sodium in the lumen or when
some other substance (e.g., choline, tetramethylammonium,
methylsulfate, sucrose) replaces sodium, chloride or both in
the lumen and the peritubular fluid simultaneously (390).
Also, reabsorption in these studies is not influenced by the
buffer used and, even when sodium is present, inhibition of
Na+ -K+ -ATPase does not disrupt reabsorption (390, 391).
Also, when sodium is present, about 25% of the net fluid
reabsorption in the proximal tubule depends on colloid in
the peritubular fluid (390). Increases in cell membrane area,
which occur within minutes following simultaneous substitutions for sodium in the luminal and peritubular fluid, suggest
that under these circumstances, colloid osmotic pressure in
the peritubular fluid or perhaps some small, previously unimportant driving force could account for maintaining reabsorption at the control level (394). However, the exact mechanism
involved in fluid reabsorption in these proximal tubules is not
yet understood and offers an excellent opportunity for studies
on what may be a unique epithelial transport process.
Water secretion. Clearance studies in sea snakes (Aipysurus laevis) suggest that net secretion of fluid at very low
levels may sometimes occur when whole-kidney GFR is very
low (2055). The tubule site of this fluid secretion and the
mechanism involved in it are unknown, but, as in fish proximal tubules and mammalian collecting ducts (1926), the
process appears to be dependent on the secretion of sodium
and chloride (2055). Whatever the mechanism, fluid secretion
certainly would appear to be necessary for any aglomerular
nephrons in reptiles to actually function as excretory organs.
Dilution and concentration. In addition to varying GFR in
response to changes in hydration (see above), many reptiles—
but not all—vary the osmolality of ureteral urine from isosmotic to the plasma to significantly hypoosmotic to the plasma
in response to changes in hydration (Table 15). These variations in osmolality of the ureteral urine, which reflect variations that actually occur along the renal tubules, are the principle means by which the kidneys alter the amount of solute-free
water delivered to the colon, cloaca or bladder (see below).
Production of tubule fluid hypoosmotic to the plasma from the
isosmotic glomerular filtrate requires reabsorption of solutes,
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Osmoregulation and Excretion
Table 15
Comprehensive Physiology
Examples of Range of Osmolal Urine-to-Plasma Ratios (U/P)
Osmolal U/P (approximate
maximum range)
References
0.55-0.95
(1629)
Desert tortoise
Gopherus agassizii (arid, terrestrial)
0.3-0.7
(395)
Freshwater turtle
Pseudemys scripta (freshwater, semiaquatic)
0.3-1.0
(395)
Horned lizard
Phrynosoma cornutum (arid, terrestrial)
0.8-1.0
(1562)
Blue spiny lizard
Sceloporus cyanogenys (arid, terrestrial)
0.3-0.7
(1776)
Sand goanna
Varanus gouldii (arid, semiaquatic)
0.4-1.0
(178)
Bull snake
Pituophis melanoleucus (arid, terrestrial)
0.5-1.0
(959)
Freshwater snake
Nerodia sipedon (freshwater, semiaquatic)
0.1-1.0
(381)
Olive sea snake
Aipysurus laevis (marine, aquatic)
0.8-1.2
(2055)
Crcodilia
Crocodile
Crocodylus acutus (freshwater and marine, semiaquatic)
Testudinea
Squamata
Sauria
Ophidia
The values are from measurements on ureteral urine. Modified from Dantzler (382) with permission.
primarily sodium and chloride, without accompanying water
somewhere along the nephrons. The tubule sites of this dilution process in reptiles have generally not been well defined.
However, micropuncture studies on a xerophilic lizard (Sceloporus cyanogenous) (1776) indicate that dilution can occur
at least by the early distal tubule and that it can continue
throughout the collecting duct. Preliminary microperfusions
of snake (Thamnophis spp.) renal tubules suggest that dilution may occur in the thin intermediate segment between the
proximal and distal tubules (S. D. Yokota and W. H. Dantzler,
unpubl. observations), and, as noted above, the distal tubules
of these animals may be specialized to permit additional dilution by sodium reabsorption from tubule fluid that is already
highly dilute (117, 119, 122).
The degree to which the osmolality of the tubule fluid can
be reduced relative to the plasma depends, not just on the magnitude of solute reabsorption, but also on the degree to which
the tubule epithelium remains impermeable to water. Freshwater reptilian species with a major need to excrete excess
water generally produce the most dilute tubule fluid (about
one-tenth the osmolality of the plasma) (Table 15). Increases
in ureteral urine osmolality from hypoosmotic toward isosmotic then reflect increases in the permeability of the tubules
to water, apparently as a result of the interaction of AVT
492
with V2 -type receptors in the tubules. Clearance studies support this regulatory role of AVT in freshwater snakes (Nerodia = Natrix sipedon) (381), freshwater turtles (Chrysemys
picta belli and Pseudemys scripta) (231, 395) and arid-living
lizards (Ctenophorus ornatus and Varanus gouldii) (176,
178). However, not all species show a wide range in variation
in osmolality of the ureteral urine (Table 15). For example,
a few species (horned lizard, Phrynosoma cornutum; olive
sea snake, Aipysurus laevis) always produce ureteral urine
close to the osmolality of the plasma, whereas others (desert
tortoise, Gopherus agassizii; blue spiny lizard, Sceloporus
cyanogenys) always produce ureteral urine hypoosmotic to
the plasma. Indeed, the micropuncture studies on Sceloporus
cyanogenys show no change in dilution along the renal tubules
in response to the administration of AVT. Ureteral urine nearly
isosmotic to the plasma always produced by horned lizards
and olive sea snakes (Table 15) appears appropriate for these
species, which rarely have excess water to excrete. However,
ureteral urine being always hypoosmotic to the plasma produced by desert tortoises and blue spiny lizards (Table 15)
appears inappropriate, for these species also rarely have
excess water to excrete. Further modification of the urine in
these latter two species clearly occurs in the bladder or cloaca
(see below).
Volume 4, April 2014
Comprehensive Physiology
Table 16
Osmoregulation and Excretion
Approximate Percentages of Total Urinary Nitrogen as Urates, Urea and Ammonia
Percent of total urinary nitrogen as
Urates
Urea
Ammonia
References
70
0-5
25
(917)
Wholly aquatic
5
20-25
20-25
(1259)
Semiaquatic
5
40-60
6-15
(82)
Mesic environment
7
30
6
Xeric environment
50-60
10-20
5
Desert tortoise
Gopherus agassizii
20-25
15-50
3-8
Freshwater turtle
Pseudemys scripta
1-24
45-95
4-44
Sauria
90
0-8
insignificant to ?
highly significant
Ophidia
98
0-2
insignificant to ?
highly significant
65-80
10-28
3-4
Crocodilia
Testudinea
Wholly terrestrial
(395)
Squamata
(382)
Rhynchocephalia
Sphenodon punctatum
(751)
Modified from Dantzler (382) with permission.
Reptiles are not capable of producing urine sufficiently
more concentrated than the plasma to be physiologically significant in the conservation of water. However, a few species
(marine snake, Aipysurus laevis; lizard, Amphibolurus maculosus; sea turtle, Chelonia mydas) can produce urine slightly
hyperosmotic to the plasma (maximum osmolal urine-toplasma ratio is about 1.2-1.3) (194, 1479, 2055). In the studies
on Chelonia mydas and Amphibolurus maculosus, urine was
not collected directly from the ureters, and thus the slightly
hyperosmotic urine obtained may actually reflect modification of ureteral urine in the bladder or cloaca. In the case of
Aipysurus laevis, the slightly hyperosmotic urine may involve
tubule secretion of solutes (e.g., sodium, potassium, magnesium or ammonium) into a very small volume of tubule fluid
(2055). Although secretion of ions in these animals may be
important for their excretion, the production of urine slightly
hyperosomotic to the plasma cannot be of physiological significance because the plasma osmolality is far below that of
sea water.
Excretory End Products of Nitrogen Metabolism:
Ammonia, Urea and Uric Acid
Ammonia. Ammonia is both highly soluble and highly toxic.
Thus, it becomes the primary excretory end product of
nitrogen metabolism only in completely aquatic vertebrate
Volume 4, April 2014
species, where it can rapidly be carried away (189). In most
vertebrate species, its primary role involves renal excretion of
acid and maintenance of acid-base balance. At present, there
is no information on the role of ammonia excretion in acidbase balance in reptiles. Moreover, ammonia has not been
found to be the primary renal means of excreting nitrogen
even in completely aquatic reptile species (189). Nevertheless, ammonia is excreted as an important end product of
nitrogen metabolism in many reptile species (Table 16) (189).
For example, in crocodilians such as Alligator mississippiensis, about 25% of the total nitrogen excreted is in the form of
ammonia and about 75% is in the form of urates (Table 16)
(387, 917). Moreover, in alligators on a standard meat diet,
the absolute amount of ammonia excreted is greater than that
for any other vertebrate studied (343, 926). Much nitrogen is
also excreted as ammonia in semiaquatic and aquatic chelonians (e.g., freshwater turtle, Pseudemys scripta) (Table 16)
(387, 395). In these animals, nitrogen excretion is frequently
distributed about equally between ammonia and urea but there
is much variation and urea tends to predominate (Table 16)
(395).
No one has measured any significant amount of ammonia in the systemic blood of reptiles. Therefore, the ammonia
present in the urine cannot come from filtration and must be
produced by the renal tubules. The exact tubule site of such
ammonia production is unknown, but it is generally assumed
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Osmoregulation and Excretion
to involve primarily the proximal tubule, as it does in mammals. Although the mechanism of ammonia production is not
known in detail, Lemieux et al. (1031), studying alligator
renal tubules in vitro, found evidence for its production from
both glutamine and alanine probably by the same enzymatic
deamination steps as in mammals. Interestingly, when alligators are dehydrated, renal ammonia production decreases and
more nitrogen is excreted in the urine in the form of urates
(926). King and Goldstein (926) suggest that during dehydration, decreased renal blood flow could decrease delivery of
glutamine and alanine to the kidneys, and that accumulation
of ammonia in renal tissue could drive the reversible deamination reactions in the direction of amino acid formation, but
these hypotheses have not been examined directly.
No experimental data are available on the mechanism by
which ammonia produced in the renal tubule cells is secreted
into the tubule lumen in reptiles. In those species in which
the pH of the lumen is below that of the cells, secretion could
occur by non-ionic diffusion of free ammonia (NH3 ) across
the luminal cell membrane followed by trapping of ammonium ion (NH4 + ) in the lumen. However, carrier-mediated
secretion of NH4 + would certainly need to occur in alligators
in which the urine and, therefore, the tubule fluid are highly
alkaline with much bicarbonate (341, 342). This might involve
the substitution of NH4 + for H+ on a Na+ /H+ exchanger on
the tubule luminal membrane.
Urea. Urea appears to be the primary form of nitrogen excretion only in some chelonian species from aquatic,
semiaquatic and mesic habitats (Table 16). However, it is a
highly important form of nitrogen excretion even in chelonian reptiles from arid terrestrial environments and also in the
one extant rhynchocephalian species (Sphenodon punctatum)
(Table 16). Urea is excreted primarily by filtration and tubular reabsorption, with the latter increasing with dehydration
and decreasing with hydration. Indeed, net tubular secretion
is observed in a few lizard species (Lacerta viridis and Sceloporus cyanogenys) and Sphenodon during extreme diuresis
with hydration (382, 1431, 1627). The tubule sites and mechanism of tubule transport have not been examined, although
both reabsorption and secretion almost certainly involve some
form of carrier-mediated process.
Urate. Urate is the primary urinary form in which excess
nitrogen is eliminated in all reptile species studied except
for some aquatic, semiaquatic and mesic chelonian species
(Table 16). The low solubility of both uric acid itself (0.384
mmol/l) and its salts (e.g., 6.76 and 12.06 mmol/l, for sodium
urate and potassium urate, respectively) (674) mean that
almost no uric acid and only modest amounts of urate salts
can actually be dissolved in the aqueous phase of the urine.
The primary cation in these urate salts that are dissolved, usually either sodium or potassium, is determined by whether
the animal is a carnivore or herbivore. However, in alligators,
which excrete large amounts of ammonium in an alkaline
urine, ammonium urate (solubility: 3.21 mmol/l) may be the
dominant salt (341, 342). In some chelonian species the concentrations of urate salts excreted may be sufficiently low that
494
Comprehensive Physiology
they all are in true aqueous solution (395). However, in many
other reptilian species the concentrations of urates are too
high to exist solely in simple solutions. Instead, the aqueous
phase of the urine probably contains most urates in the form
of relatively small lyophobic colloid particles and much larger
lyophilic colloids (1232, 1470).
Most urate excreted by reptiles actually is in the form of
precipitates, not only in the bladder or cloaca, where precipitation might be expected to occur, but also in the collecting
ducts and ureters (1237) (W. H. Dantzler, unpublished observations). In the collecting ducts and ureters, these precipitates
are in the form of small, smooth spheres of unknown chemical structure, which should pass along the these tubular structures without causing damage; in the bladder or cloaca of the
same species, they are in the form of sharp-edged crystals,
which may have formed only in those regions, as well as the
small spheres (1237). These precipitates, whatever their exact
chemical structure, contain significant amounts of inorganic
cations, largely sodium or potassium, but sometimes calcium
or magnesium (387, 1234). In alligators, they may contain
ammonium (341, 342). The exact cations in the precipitates
depend on the acid-base status of the animal as well as on
dietary intake (387, 1234). In any case, the cations combined
with these precipitates are excreted without water and do not
contribute to the osmolality of the urine. Therefore, the osmolality of the urine in reptiles does not reflect the ability of these
animals to excrete inorganic ions. In addition, in the colon,
cloaca or bladder, less complex precipitates may form and
urate salts may be converted to uric acid, particularly during the acidification observed in bladders of some turtles and
cloacae of some lizards (640, 1236, 1760). This process can
free up cations for further reabsorption, if required.
In all reptiles studied, the urate in the plasma is freely filtered (152) and net secreted by the renal tubules (382). Almost
all the information available on the tubule transport process
comes from in vitro microperfusions of snake (Thamnophis
spp.) renal tubules. These studies indicate that transport from
peritubular fluid to luminal fluid occurs against a concentration gradient throughout the proximal tubule but not in the
distal tubule (375, 378). There is no net reabsorption, but there
is a significant passive unidirectional backflux throughout the
proximal tubule (Fig. 30) (375).
Net secretion in these snake tubules involves transport
into the cells against an electrochemical gradient at the basolateral side and movement from the cells into the lumen
down an electrochemical gradient (375). The step at the
basolateral membrane appears to involve sodium-independent
countertransport for another unknown anion (Fig. 30) (278,
392, 1260, 1510). This step is completely separate from
that for other commonly secreted organic anions (e.g. paraaminohippurate) (278, 382, 392). So far, there is no convincing evidence from studies on these isolated snake tubules or
on brush border membrane vesicles from snake kidneys that
the movement of urate from the cells to the tubule lumen is
mediated in any way (Fig. 30) (107, 389, 1260). All the data
are compatible with simple passive diffusion, but, given the
Volume 4, April 2014
Comprehensive Physiology
0.5mV
Osmoregulation and Excretion
–60mV
Urate–
0mV
Urate–
?
A–
PL = 0.75 ×
cm ⋅ s–1
10–5
PP = 3.10 × 10–5
cm ⋅ s–1
Urate– ?
Urate
Jbl
Kt ≅ 150 μM
Jmax ≅ 140 fmol min–1 mm–1
the transport system is normally saturated (or nearly saturated) (382). This suggests that changes in plasma urate concentration have little effect on net urate secretion. Instead,
net secretion (and thus excretion) appears to depend much
more on flow rate through the lumen and backdiffusion (382,
392). Given the low solubility of urate, these characteristics of urate transport may be very important in relation to
changes in the number of filtering nephrons during dehydration and hydration. The relatively high passive permeability
of the basolateral cell membrane, the apparent dependence
of transport into the cells across the basolateral membrane
on the presence of flow along the lumen, and the apparent
large paracellular backleak during low luminal flow rates all
may function together to prevent the accumulation of urate
in the cells or tubule lumens of nephrons that are not filtering (382, 392). In turn, these transport characteristics may
enhance net urate secretion when most (or all) nephrons
are filtering and flow along the nephron lumens is high
(382, 392).
Figure 30
Bladder, Cloaca and Colon
large urate flux across this membrane during net secretion,
simple passive diffusion appears highly unlikely (392).
There are a number of other unusual features of transepithelial urate transport in snake renal tubules, which taken
together may have adaptive significance (392). First, the
apparent passive permeability of the basolateral membrane
(3.10 × 10−5 cm/s) is more than four times that of the luminal membrane (0.75 × 10−5 cm/s) so that transport into
the cells against an electrochemical gradient at the basolateral membrane is always working against a significant
backleak (Fig. 30) (393). Second, function of the basolateral transport step appears to require filtrate (or an artificial
perfusate) flowing along the tubule lumen (375). Third, net
transepithelial secretion varies directly with the rate of luminal perfusion, reflecting the large transepithelial backdiffusion at low perfusion rates (375, 383). Moreover, the passive transepithelial permeability determined experimentally
from this transepithelial backdiffusion (2.4 × 10−5 cm/s)
(375, 378) is about four times the value calculated from
the independently measured individual membrane permeabilities (0.60 × 10−5 cm/s), indicating that much of this backdiffusion occurs by a paracellular route (378). Finally, the
apparent Kt measured for net transepithelial secretion (about
150 μmol/l) (Fig. 30) is significantly below the normal plasma
urate concentration (about 400-500 μmol/l), indicating that
The water and electrolyte composition of ureteral urine is
modified before final excretion by structures distal to the kidneys. The exact combination of the structures involved (bladder, cloaca and colon) depends on the species. No ophidians
or crocodilians have urinary bladders, whereas all chelonians have them. Urinary bladders are also found in a number
of lizards and in the one extant rhynchocephalian (Sphenodon punctatum). However, studies on water and ion reabsorption by reptilian bladders are far from comprehensive
and give variable results. Turtle (Pseudymys scripta) and tortoise (Gopherus agassizii and Testudo graeca) bladders show
significant osmotic water permeability both in vivo and in
vitro (97, 203, 204, 395). However, they do not respond to
antidiuretic hormone, at least in vitro (97) (W. H. Dantzler,
unpublished obsv.). Nevertheless, the bladder of the desert
tortoise (Gopherus agassizii) serves as an important organ for
storing water during the dry seasons (856, 1285), as well as a
sink for potassium, urea and urates (1233). Indeed, masses
of precipitated urates appear to be stored in the bladder
of these animals and expelled with large volumes of water
when free water is plentiful (W. H. Dantzler, unpublished
obsv.). The urinary bladder of turtles also shows significant
aldosterone-stimulated sodium reabsorption in vitro, a feature
that enhances water reabsorption and should be important
in post-renal regulation of excretion (97, 204, 1029). However, it should be noted that the bladder of the desert tortoise
(Gopherus agassizii) has a well-developed sphincter, which
not only keeps water tightly held within the bladder but also
permits ureteral urine to bypass it and move directly into the
cloaca (395) (W. H. Dantzler, unpublished obsv.). This sphincter is absent in freshwater turtles (Pseudemys scripta) (395)
(W. H. Dantzler, unpublished obsv.). These features certainly
help determine the degree to which the bladder and cloaca
alter the composition of the ureteral urine.
Model for net tubular secretion of urate based on studies with snake (Thamnophis spp.) proximal renal tubules. Open circle
with solid arrow indicates either primary or secondary active transport. For countertransport, solid arrow indicates movement against
electrochemical gradient, and broken arrow indicates movement down
electrochemical gradient. Broken arrows with question marks indicate
possible passive movements. A− indicates anion of unspecified nature.
Apparent permeabilities of luminal (PL ) and peritubular (PP ) membranes
are shown. Apparent Kt and Jmax for net secretion are shown at bottom of the figure. Reproduced from Dantzler and Bradshaw (392) with
permission.
Volume 4, April 2014
495
Osmoregulation and Excretion
The information about chelonian bladder function,
although far from comprehensive, is much more extensive
than that available for Sphenodon or for those lizards that
have them. The bladder of the large skink (Tiliqua rugosa)
has been reported to be water permeable in vitro (94), but
there is no significant information on its function in vivo. In
the Rhynchocephalian (Sphenodon punctatum), no net water
movement across the wall of the bladder was observed in
vivo (1627). In some lizards with bladders (e.g., Hemidactylus fluviatilis), there is a well-developed bladder sphincter
(1651) like that in the desert tortoise and ureteral urine may
frequently bypass the bladder (392).
Cloacal and colonic function in modification of ureteral
urine and, thus, maintenance of water and electrolyte balance has been studied in some detail only in lizards and, to
a lesser extent, in crocodilians (392), although an early study
indicated that one species of snake (Xenodon sp.) would die
within a few days if ureteral urine was not allowed to enter
the cloacal-colonic complex (859). A recent comparative morphological and histochemical study of the colon and cloaca of
water snakes from marine (Nerodia clarkia clarkia) and freshwater (N. fasciata) habitats showed no differences between
the two species in cell structure or in the distribution of Na+ K− -ATPase and Na+ -K+ -2Cl− cotransporter (57). These data
suggest that whatever role these factors play in colon or cloacal modification of ureteral urine, they do not contribute to
N. clarkia clarkia to function in a marine habitat.
A number of investigators have compared ureteral urine
with voided urine in lizards and crocodilians and noted that
significant amounts of electrolytes and water were apparently reabsorbed from the cloacal-colon complex (174, 176,
1629). For example, Bradshaw (174, 176) found that in the
lizard Ctenophorus (= Amphibolurus) ornatus, the relative
osmolar clearance (COSM /CIN ) fell from 29% in the ureteral
urine to 1% in the voided urine. From a number of such
studies on lizards, Bradshaw (173, 176) concluded that the
cloacal-colonic complex reabsorbs about 90% of the fluid presented to it during either a water or saline dieresis. However,
sodium reabsorption from the fluid presented to the cloacalcolonic complex decreased from about 99% during a water
diuresis to about 35% during a saline diuresis, indicating
some form of regulation. Bradshaw and Rice (178) reached
a similar conclusion in studies on conscious unanesthetized
lizards (Varanus gouldii), held at their preferred body temperature, in which they perfused the cloacal-colonic complex
in vivo et situ with artificial ureteral urine at rates appropriate for delivery of ureteral urine during hydration, dehydration and salt loading. During salt loading, compared with
hydration, water reabsorption increased significantly from
∼23% to ∼40% and sodium reabsorption decreased significantly from ∼34% to ∼22%. The response to dehydration was essentially the same as during salt loading, with an
increased rate of reabsorption of water and a decreased rate
of reabsorption of sodium. When Bradshaw and Rice (178)
calculated the electrolyte concentrations of the reabsorbate
with these treatments, they found that it was hypoosmotic
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Comprehensive Physiology
with both dehydration and salt loading and hyperosmotic
with water loading, suggesting solute-linked water reabsorption as indicated earlier for crocodiles (Crocodylus acutus)
(1629).
The mechanisms involved in the observed water and
electrolyte reabsorption from the cloacal-colonic complex
in reptiles are not well understood. A mecholyl-inhibitable,
mucosa-negative transepithelial potential has been recorded
across cloaca-colon preparations in a number of snakes,
lizards, a tortoise and a crocodilian (97, 99, 104, 859, 1620).
This observation is compatible with active sodium transport
from the lumen side (mucosa) to the blood side (serosa) of
these preparations and suggests that transport of water is
coupled to sodium transport, as demonstrated by Bindslev
and Skadhauge (137) in the large intestine and coprodium
of chickens. However, the details of coupling under different reabsorptive conditions have not been examined. Finally,
water reabsorption can be facilitated by differences in colloid osmotic pressure across the cloaca of the desert iguana
(Dipsosaurus dorsalis), and presumably this could occur in
other species.
Salt Glands
Many reptile species have specialized extrarenal glands capable of excreting hyperosmotic salt solutions, thereby contributing to the elimination of excess electrolytes (primarily sodium or potassium) while conserving free water. These
glands may be external nasal glands, lachrymal glands or
premaxillary, sublingual or lingual glands, depending on the
species (392). Although salt glands are present in many
species, they have not been found in terrestrial snakes, alligators (Alligator mississippiensis), geckoes and the one species
of pygopodid lizard that has been studied (392, 1595). To
date, they have only been described in one species of terrestrial chelonian (Testudo carbonaria) (1410).
The primary cation secreted by reptilian salt glands is
clearly related to diet and habitat (see table 10.13 in Dantzler and Bradshaw [392] for a detailed review). Regardless
of anatomical site or embryological origin, the salt glands
of marine species secrete sodium as the primary cation, frequently at extremely high rates that can vary with the salinity
of the water to which they are adapted (356), whereas the
salt glands of terrestrial tortoises and lizards generally secrete
potassium as the primary cation, reflecting their herbivorous
diet (392). There are some exceptions. The salt glands of
two terrestrial varanid lizards (Varanus semiremex and V. salvator), which eat crustaceans and other animals in marine
mangrove swamps and take in much salt water, secrete primarily sodium (392, 463, 1235). Of perhaps more interest is
the observation that the salt glands of the Galapagos terrestrial iguana, which eat a herbivorous diet primarily of Opuntia (prickly pear) cactus, secrete substantially more sodium
than potassium (461). Also, the salt glands of some terrestrial lizards (e.g., desert iguana Dipsosaurus dorsalis), while
normally secreting potassium as the primary cation, can be
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Comprehensive Physiology
stimulated to secrete very large amounts of sodium and even
rubidium (1677).
Although less is known about the detailed structure, especially ultrastructure, of reptilian salt glands than of avian salt
glands (792), the glands are known to consist of a series of
secretory tubules that empty into a duct (392). In those salt
glands studied, the cells of the tubule epithelium are least
specialized at the blind end of the tubule. They become progressively more differentiated into mitochondrion-rich principal cells with extensive lateral membrane infoldings along
the length of the tubule away from the blind end (392).
In some species (e.g., Iguana iguana and Tiliqua rugosa),
mucoserous cells are interspersed among the principal cells
(392). Because of the many mitochondria and the presence
of extensive Na+ -K+ -ATPase along the lateral infoldings in
the principal cells (490), they are considered to be the cells
involved in electrolyte secretion (392). Bumetanide (a blocker
of the Na+ -2Cl− -K+ -cotransporter) and ouabain (a blocker
of Na+ -K+ -ATPase) both inhibit sodium secretion by isolated salt glands from Malaclemys terrapin (1685). These
observations suggest that, as in avian salt glands, the secretory process in principal cells in those reptilian salt glands
secreting primarily sodium involves these two transporters.
Apparently, chloride enters the principal cells at the basolateral membrane via secondary active transport involving the
Na+ -2Cl− -K+ -cotransporter and then moves into the lumen
through chloride channels with sodium accompanying it via
a paracellular pathway. The energy for the process derives
from the Na+ -K+ -ATPase (1684), the amount of which can
increase with adaptation to increased secretion (354) (see section on Aves for more detail on the secretory process). This
transport can be stimulated by cholinergic activation and by
activation of the adenylate cyclase pathway by a number of
activators, including vasoactive intestinal peptide and possibly other peptides (e.g., B-type natriuretic peptide) (353,
355, 549, 1684, 1685). There are not yet any definitive data
on mechanism by which the sodium concentration increases
to significantly hyperosmotic levels along the length of the
secretory tubules (392).
The mechanism by which secretion occurs in those reptilian salt glands in which potassium is the primary ion secreted
is not at all understood. Studies by Shuttleworth et al. (1686)
on potassium secretion by nasal salt glands in the desert lizard
Sauromalus obesus show that stimulation of secretion can
lead to a doubling of Na+ -K+ -ATPase activity without any
increase in the size or weight of the gland, and that as much
as 68% of the potassium in fluid perfusing the gland can be
extracted in a single passage. Indeed, Shuttleworth and Hildebrandt (1684) suggest that production of a secretion containing potassium in excess of 1,000 mmol/l with essentially no
sodium in this species and others must require a unique mechanism quite different from that for the sodium secreting glands
in either reptiles or birds. Whatever the mechanisms involved,
salt glands capable of secreting very high concentrations of
either sodium or potassium certainly play an important role in
regulating the internal environment of many reptile species.
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Osmoregulation and Excretion
Aves
Birds successfully occupy all habitats on earth, and, like mammals, their water and electrolyte milieu intérieur is relatively
tightly controlled. To do this, they have evolved a number
of physiological mechanisms to assist in osmoregulation and
excretion. These mechanisms involve the kidneys, lower gastrointestinal tract and nasal salt glands, which are considered
in that order in this section.
Function and Regulation of the Physiological
Mechanisms for Osmoregulation
Kidneys
The kidneys play a critical role in regulating the composition
of the internal environment of birds, as they do in other tetrapod vertebrates, by controlling the excretion of water, ions and
nitrogenous wastes. However, in all birds, substantial modification of the urine occurs distal to the kidneys in the lower
gastrointestinal tract. Moreover, in marine species, nasal salt
glands contribute to the excretion of ions (primarily sodium)
in excess of water. Nevertheless, the kidneys are quantitatively
very important in regulating the output of ions and water and,
therefore, the composition of the internal environment. This
regulation involves adjustments in both filtration at the renal
glomerulus and in reabsorption and secretion along the renal
tubules.
General form of kidneys and nephrons
The kidneys of birds vary less among species in external
gross appearance than those of amphibians, reptiles and even
mammals. In all species, the paired kidneys are prominent
retroperitoneal organs, which are flattened and recessed into
the bony synsacrum (fused lumbar, sacral and caudal vertebrae). They are crossed by the major blood vessels and nerve
trunks, binding them tightly into this bony concavity. Each
kidney is divided into three divisions—cranial, medial, and
caudal—which, depending on the species, may or may not be
obvious superficially (853).
The avian kidney consists of cortical and medullary zones,
but they are not as clearly demarcated as in mammals. Moreover, it contains two general types of nephrons. The most
numerous type (e.g., about 90% in Gambel’s quail, Callipepla
gambelii; about 70% in European starlings, Sturnus vulgaris)
is short, lacks a loop of Henle and is often referred to as a
“reptilian-type” or “loopless” nephron. These are found in the
superficial or cortical region of the kidney (Fig. 31). These
nephrons, which are simpler than true reptilian nephrons are,
consist of a small glomerulus, no neck segment, a proximal tubule with a few simple folds, no intermediate segment and a distal tubule with only one main fold before it
empties at right angles into a collecting duct (Fig. 31) (187,
1988). In some of these nephrons, there is a short connecting segment between the distal tubule and the collecting duct
497
Osmoregulation and Excretion
Comprehensive Physiology
Afferent vein
Collecting duct
Central vein (efferent)
Arterial supply
Reptilian-type nephron
Capillary plexus
Short-loop mammalian-type nephron
Long-loop mammalian-type nephron
0 1
mm
0 0.1
mm
Medullary cone
Ureteral branch
0 10
mm
Ureter
Figure 31 Illustration of detailed internal organization of avian kidney. The whole kidney is shown at the lower left
with two successive enlargements of sections of the kidney showing increasing internal detail. The largest section shown
gives detail of nephrons forming a single medullary cone. Near the surface of the kidney are the small loopless reptiliantype nephrons arranged in a radiating pattern around a central vein to form a single lobule. In the deeper regions of
the cortex are the larger mammalian-type nephrons with highly convoluted proximal tubules, loops of Henle and distal
tubules. A gradual transition occurs from loopless reptilian-type nephrons to longest-looped, mammalian-type nephrons.
The loops of Henle are in parallel with collecting ducts and vasa recta within a medullary cone, an arrangement that allows
countercurrent multiplication (see text). Reproduced from Braun and Dantzler (187) with permission.
(1988). The second, less numerous nephron type (about 10%
in Gambel’s quail; about 30% in starlings) is located deep
in the cortex and extends into the medulla (Fig. 31). These
nephrons have loops of Henle, resemble short-looped mammalian nephrons and are referred to as “mammalian-type” or
“looped” nephrons. They consist of a glomerulus, which is
larger than that of the reptilian-type nephrons, a convoluted
proximal tubule, a straight proximal tubule, thin descending limb of Henle’s loop, a thick ascending limb of Henle’s
loop that always begins on the descending side before the
bend (prebend region), a distal convoluted tubule and possibly a short terminal connecting tubule before they enter
collecting ducts (Fig. 31). It is important to note, however,
that the transition from the most superficial reptilian-type to
the mammalian-type nephrons is gradual so that there are
nephrons of intermediate size and intermediate loop length
in the region between the superficial cortical lobules and the
deeper medullary region. In all nephrons, the initial portion
of the distal tubule in each nephron appears to be tightly
apposed to the vascular pole of its own glomerulus, as it
is in amphibians, reptiles and mammals. Renin-producing
498
granular cells are found in the glomerular arterioles of all four
of these vertebrate classes. However, avian nephrons, in contrast to amphibian and reptilian nephrons and like mammalian
nephrons, also appear to have macula densa cells (1250, 1726)
and, thus, possibly a complete juxtaglomerular apparatus.
The loops of Henle of the mammalian-type nephrons, vasa
recta, which arise from the efferent arterioles of glomeruli of
these nephrons, and the collecting ducts, which drain both
the reptilian-type and the mammalian-type nephrons from a
single radial group of cortical lobules, are arranged in parallel
and bound together by a connective tissue sheath into a tapering structure referred to as a medullary cone (Fig. 31). As in
the mammalian kidney, the parallel arrangement of loops of
Henle, vasa recta and collecting ducts suggests the capability
of producing urine hyperosmotic to the plasma (see below).
Indeed, the gross structural features of the loops and collecting ducts and their arrangement in the medullary cones are
similar to the outer medulla of the developing neonatal rat
kidney (1082). However, unlike the mammalian kidney, there
is no renal pelvis. The collecting ducts coalesce within this
connective tissue sheath to form a single branch of the ureter
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Comprehensive Physiology
(Fig. 31). The number of medullary cones, and thus the number of organized units of the kidney (see Fig. 31 for a single
unit) per kidney, is relatively constant for a given species but
varies markedly among species (853).
Like amphibian and reptilian kidneys, avian kidneys have
both arterial and renal portal blood supplies. The extent to
which venous portal blood supplies the kidney is determined
by a smooth muscle valve located in the external iliac vein,
which is under autonomic nervous control (227). When the
valve is closed, portal venous blood contributes to the peritubular capillary blood supply of the reptilian-type nephrons
in the cortical region of the kidney (no venous portal blood
enters the medullary cones); when the valve is open, venous
portal blood bypasses the kidney.
Glomerular function
As in glomerular nephrons of other vertebrates, the initial process in urine formation is the production of an ultrafiltrate of
the plasma at the renal glomerulus. This ultrafiltrate supplies
water and small solutes to the proximal tubule in the same
concentrations as in the plasma. As in other vertebrates, the
forces involved in this process are assumed to be the same
as those described in detail for reptiles (see above). However,
there have been no direct measurements of pressures within
the avian kidney. But because birds, like mammals, maintain a high stable arterial blood pressure, the pressures at the
glomerulus may be similar to those found in mammals.
However, the capillary network within avian glomeruli
is much simpler than that in mammalian glomeruli and the
diameter of the avian glomerular capillaries is twice that of
mammalian ones (183). Indeed, the network is simpler than
that found in reptiles (2056). The glomerular network of the
small, loopless, reptilian-type avian nephrons consists of only
a couple of loops of a single unbranched capillary (183). The
glomerular network of the larger, looped, mammalian-type
nephrons consists of a few more loops and may have one
or two connecting branches, very different from the highly
branched network in mammalian glomeruli (183). Braun
(183) suggests that the simple network of capillaries with
large diameters is related to the large, nucleated, rigid, nondeformable avian red blood cells.
There is no information on whether this simple network
has any direct effect on the factors involved in the filtration
process. However, it seems likely that, although the glomerular capillary diameter is larger than in mammals, the much
simpler capillary network would actually make the surface
area available for filtration smaller than in mammals. It should
also be noted that the filtration barrier appears to be less
restrictive to large molecules in avian glomeruli than in mammals because large amounts of protein (about 5 mg/ml) appear
in the ureteral urine of birds (183). This greater glomerular permeability in birds than in mammals may result from
larger slit pores between podocytes and a smaller negative
charge on the glycocalyx of the filtration barrier in avian
glomeruli than in mammalian glomeruli (259). The apparent
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Osmoregulation and Excretion
physiological significance of protein in the urine is discussed
below.
SNGFRs for both reptilian-type and mammalian-type
nephrons in birds and superficial and juxtamedullary nephrons
in mammals have been measured by the constant infusion
sodium ferrocyanide precipitation technique, and the most
superficial nephrons in birds and mammals have been measured by the micropuncture technique (Table 17) (184, 187,
196, 382, 1015, 1865). As shown in Table 1, the average SNGFR values for reptilian-type and mammalian-type
nephrons are essentially the same in both European starlings
and Gambel’s quail, and the average value for mammaliantype nephrons is twice that for reptilian-type nephrons. However, the average SNGFR values for the smallest, most superficial reptilian-type nephrons, the only ones accessible to
micropuncture, are only about 1/20th of the average values for
reptilian-type nephrons as a group (Table 17). Values for these
smallest reptilian-type nephrons, determined by the sodium
ferrocyanide technique, are similar to those determined by
micropuncture (E. J. Braun, personal communication).
The average SNGFR for superficial nephrons in mammals, measured either by the sodium ferrocyanide precipitation technique or the micropuncture technique, is twice
that of the mammalian-type nephrons in birds (Table 17). The
SNGFR of juxtamedullary nephrons in mammals, determined
by the sodium ferrocyanide technique, is more than three
times that of the mammalian-type nephrons in birds. Because
both the mammals and birds studied have similar mean arterial pressures, it seems likely that the much lower SNGFRs in
the birds compared to the mammals reflect the smaller surface
area available for filtration in the simple glomerular capillary
network in birds. Despite the fact that the SNGFR of bird
nephrons is much less than that of mammalian nephrons, the
whole-kidney GFR of birds is essentially the same as that of
mammals of the same body mass because bird kidneys have
many more nephrons than mammalian kidneys (240).
The whole-kidney GFR in birds increases with a water
load and decreases with a salt load or dehydration (186,
187, 386, 1561, 1703). Measurements of SNGFRs in Gambel’s quail indicate that the decrease in whole-kidney GFR
results from a decrease in the number of filtering reptiliantype nephrons with no change in the SNGFR of those filtering
(187). At this time, all mammalian-type nephrons continue filtering at the control rates (187). Further studies indicate that
this decrease in number of filtering nephrons can be duplicated
by physiological doses of the natural avian antidiuretic hormone, arginine vasotocin (AVT), and that the effect involves
constriction of afferent arterioles of the nephrons that cease
filtering (182, 185). This decrease in the number of filtering
reptilian-type nephrons, as in reptiles, can help conserve water
at the expense of excreting waste products. It appears to be
a reasonable physiological response to dehydration because
these nephrons are not arranged to function in concert to
contribute directly to the production of hyperosmotic urine.
Moreover, as in reptiles, the renal tubules of the reptiliantype nephrons that have ceased filtering can continue to be
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Osmoregulation and Excretion
Table 17
Comprehensive Physiology
Examples of Single Nephron Glomerular Filtration Rates (SNGFR) (1264)
Species and Habitat
Nephron Type
SNGFR
nl min−1
References
Mammalian-type
14.6±0.79 (27)a
(187)
Reptilian-type
6.4±0.25 (41)a
(187)
Birds
Galliformes
Gambel’s quail
Callipepla gambelii
Arid, terrestrial
(14)b
Smallest Reptilian-type
0.37±0.82
Mammalian-type
15.6±0.75 (207)a
(184)
Reptilian-type
7.0±0.35 (185)a
(184)
(377)
Passeriformes
European starling
Sturnus vulgaris
Moist, arboreal
(17)b
Smallest Reptilian-type
0.36±0.040
Superficial
30.1±2.55 (7)b
(196)
Superficial
36.4±3.5 (4)a
(1865)
(1015)
Mammals
Rat
Rattus norvegicus
Terrestrial
Juxtamedullary
51.7±6.7
Values are means ± SE. Figures in parentheses indicate number determinations.
ferrocyanide technique. b SNGFRs determined by micropuncture technique.
nurtured by the renal portal contribution to the peritubular
capillaries. The increase in whole-kidney GFR with a water
load reflects an increase in SNGFRs of both mammalian-type
and reptilian-type nephrons while all nephrons are filtering
(186). It appears very likely that during a normal state of
hydration not all the nephrons are filtering (382).
Regulation of glomerular filtration rates by other
endocrine systems (e.g., renin-angiotensin system) or by renal
nerves, although frequently suggested, has not been clearly
demonstrated in birds. Also, although birds appear to have
a complete juxtaglomerular apparatus, the presence of a distal tubule-glomerular feedback system has yet to be demonstrated. Nevertheless, avian kidneys exhibit autoregulation of
whole-kidney GFR over a renal arterial perfusion pressure of
60 mmHg to 120 mmHg, and the renal portal system appears
to play a role in maintaining effective renal plasma flow (1989,
1990).
Tubule function
The renal tubules modify the initial glomerular filtrate by
the processes of reabsorption and secretion, thereby determining the final volume, ionic composition and osmolality
of the ureteral urine. The following discussion is limited to
tubule transport of sodium, potassium, water and the primary
excretory end products of nitrogen metabolism.
500
a SNGFRs
(4)a
determined by constant infusion sodium
Inorganic ion transport Sodium. Clearance studies on
intact animals suggest that the renal tubules of birds, like
those of mammals, are capable of reabsorbing 99% of the filtered sodium (382, 1702, 1703). However, as noted above for
reptiles, clearance studies in birds do not include any filtered
sodium that is contained in urate precipitates (see below) and,
therefore, may actually overestimate the fraction of filtered
sodium reabsorbed by the renal tubules. In addition, clearance studies only indicate net sodium transport, not whether
both secretion and reabsorption of sodium occur. Moreover,
further sodium reabsorption may occur in the cloaca (1702).
Estimates derived from in vivo micropuncture studies and
in vitro microperfusion studies suggest that about 60-65%
of the filtered sodium can be reabsorbed over the length of
the proximal tubule (205, 1015). The micropuncture studies
show that sodium and water are reabsorbed isosmotically and
that chloride is reabsorbed at the same rate and to the same
extent as sodium (1015). No information is available on the
details of the transepithelial reabsorptive process for sodium
in avian proximal tubules. However, isosmotic fluid reabsorption in isolated perfused avian proximal tubules is inhibited
by ouabain in the peritubular bathing medium, indicating that,
as in other vertebrates, the reabsorptive process depends on
the action of Na+ -K+ -ATPase in the basolateral membrane to
transport sodium out of the cells (205).
In vitro microperfusion of isolated thick ascending limbs
from large mammalian-type nephrons of Japanese quail
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Comprehensive Physiology
(Coturnix coturnix) indicate that these segments reabsorb significant amounts of sodium and chloride without water (1240).
As in mammals, these segments have a very low osmotic
water permeability (1240) and a lumen-positive transepithelial potential (about +9 mV), which is dependent on the presence of both sodium and chloride in the lumen and which
is inhibited by furosemide in the lumen and ouabain in the
peritubular bathing medium (1317). These data suggest that
reabsorption of sodium and chloride involves entry into the
cells via the Na+ -2Cl− -K+ cotransporter in the luminal membrane and transport of sodium out of the cells via Na+ -K+ ATPase in the basolateral membrane as in the thick ascending
limb of outer medullary nephrons in the mammalian kidney
(1317). Therefore, these segments in mammalian-type avian
nephrons, like those in mammalian nephrons, appear to be
diluting segments and to function in the urine concentrating mechanism (see below). The early segment of the distal
tubule of small reptilian-type nephrons, which is in close
contact with its parent glomerulus, also has a small lumenpositive potential (1317) and may reabsorb sodium without
water in a similar manner to the thick ascending limb of the
mammalian-type nephrons, and therefore may function as a
diluting segment in reptilian-type nephrons (see below).
Nothing is known about sodium reabsorption in the late
distal tubules or collecting ducts in birds. However, the difference between the percent of filtered sodium reabsorbed in
the proximal tubule and the percent apparently excreted in
the urine suggests that, in addition to significant reabsorption
in the diluting segments, there is reabsorption in late distal
tubules and collecting ducts.
Almost nothing is known about endocrine control of
tubule sodium reabsorption in birds. AVT has no effect on
sodium reabsorption in thick ascending limbs of mammaliantype nephrons isolated and perfused in vitro (1240). However,
in ducks (Anas platyrhynchos), adrenalectomy and administration of corticosteroids suggests that aldosterone and corticosterone, both naturally occurring adrenal corticosteroids,
may have physiological roles in stimulating tubule sodium
reabsorption (791), but no direct studies on tubule transport
have been made. There have also been no studies on neural
regulation of sodium transport.
Potassium. Clearance studies on domestic fowl indicate
that about 10% to 25% of the filtered potassium is usually
excreted in ureteral urine, the percent probably influenced by
the amount of potassium in the diet (1703), and actual net
secretion may occur. However, interpretation of these clearance studies with regard to potassium transport has the same
caveats as mentioned above for sodium transport by the renal
tubules.
In vivo micropuncture studies on the proximal tubule of
the smallest reptilian-type nephrons in starlings indicate that
reabsorption of potassium generally occurs over the accessible portion of the tubule but variability in the amount of reabsorption is much greater than for sodium (1015). Indeed, some
punctures suggest that secretion may occur (1015). Secretion
of potassium along the proximal tubule would be markedly
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Osmoregulation and Excretion
different from what occurs in mammals and probably other
vertebrates, where secretion only occurs in distal nephron
segments (377). However, although net proximal tubule secretion cannot be ruled out, the small number of points indicating secretion makes definitive interpretation difficult (1015).
There are no direct studies of potassium transport along other
segments of avian nephrons, although it seems likely that
secretion can occur in distal portions of the nephrons (189).
No studies have been performed on the mechanism of potassium transport in avian nephrons.
The data on hormonal regulation of potassium transport
are few and incomplete. AVT has no effect on potassium
excretion in clearance studies (25, 167). Mineralocorticoids
like aldosterone might be expected to stimulate potassium
secretion as they do in other vertebrates studied, and indeed,
administration of aldosterone and corticosterone to ducks
does increase fractional excretion of potassium (789). However, the possible tubule sites and mechanism of action of this
effect have not been explored.
Water transport Water reabsorption. Water reabsorption
by the renal tubules plays a critical role in the overall water
balance of birds as it does in other vertebrates. Sometimes
it occurs at the same rate as solute reabsorption (as isosmotic fluid) and sometimes it lags behind, depending on
the need to conserve or excrete water. As in the case of
sodium, clearance studies indicate that 99% of the filtered
water can be reabsorbed by the renal tubules (377, 1702,
1703). However, in many circumstances a good portion of
the filtered water actually is recovered in the lower intestine
(see below).
As noted above, micropunctures of the proximal tubules
of the superficial reptilian-type nephrons indicate that sodium
and water are reabsorbed together in an isosmotic solution
(1015). Therefore, as is true for sodium (see above), 6065% of the filtered water is reabsorbed along the proximal
tubule (205, 1015). The reabsorption rate at this time is
about 2 nl min−1 mm−1 in isolated perfused proximal tubules
of transitional short-looped mammalian-type nephrons of
domestic chickens (205), whereas it is estimated to be about
0.35 nl min−1 mm−1 in the micropunctured smallest loopless
reptilian-type nephrons of starlings (1015). These differences
reflect differences in size and filtration rates of these nephron
populations (184, 187, 1015).
The isosmotic water reabsorption in avian proximal
tubules depends completely on sodium reabsorption, as
demonstrated by its inhibition by ouabain in isolated perfused proximal segments (see above) (205). Therefore, it is
influenced by anything that influences sodium reabsorption.
The details of the process by which water is coupled to sodium
reabsorption in avian proximal tubules have not been examined, but the water movement across the epithelium is certainly facilitated by the presence of the water channel aquaporin 1 (AQP1) in both the luminal and basolateral membranes
(261).
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Osmoregulation and Excretion
Table 18
Comprehensive Physiology
Examples of Range of Urine-to-Plasma (U/P) Osmolality Ratios
Species
Osmolal U/P
Environment and Mode of Existence
References
Chicken
0.1-2.0
Moist, terrestrial
(25)
Gallinaceous
(386)
Arid, terrestrial
(186, 187)
Gallus gallus domesticus
(1703)
Gambel’s quail
0.5-2.5
Callipepla gambelii
Gallinaceous
Measurements made on ureteral urine.
Dilution and concentration. Reabsorption of water
beyond the proximal tubule varies depending on the need
to excrete or conserve water. Studies on intact birds indicate
that they are all capable of producing dilute urine, the most
dilute urine (lowest urine-to-plasma osmolality ratio) generally being found in species with the most access to fresh water
(see examples in Table 18) (377). The maximum dilution
appears to be as great as that found in any other vertebrates
(377). As discussed in the section on reptiles (see above),
dilution requires the reabsorption of solute (primarily sodium
chloride) without accompanying water in some segment or
segments of the nephrons distal to the proximal tubule. As
described above, this process apparently can occur in the
thick ascending limb of Henle’s loop (including the prebend
segment) of the looped mammalian-type nephrons and possibly in the early portion of the distal tubule of small loopless
reptilian-type nephrons (1240, 1317). Whether any significant
dilution can occur beyond these regions is unknown, although
it appears very likely that the collecting ducts are capable of
performing some additional dilution (1319). In any case, all
nephrons (short- and long-looped and loopless) can contribute
to the production of urine hypoosmotic to the plasma.
Birds, like mammals and unlike all other non-mammalian
vertebrates, are capable of producing urine hyperosmotic to
the plasma, but this ability is rather limited (maximum U/P
osmolality: about 2.0-2.5) (see, for example, Table 18) (189,
377). This urine-concentrating ability is related to the presence of nephrons with loops of Henle arranged parallel to
collecting ducts and looped capillaries in the kidneys of birds
and mammals (377) and the resulting production of an increasing osmotic gradient along the length of the medullary cones
in bird kidneys (491, 1704) and the medulla in mammalian
kidneys (377). In contrast to mammalian kidneys, however,
in which this osmotic gradient involves both sodium chloride
and urea, in avian kidneys the gradient consists almost entirely
of sodium chloride and, to a very slight extent, potassium
chloride (1704). Nevertheless, as in mammalian kidneys, the
generation of this gradient appears to involve countercurrent
multiplication (377).
On the basis primarily of studies of tubule transport characteristics in Japanese quail, Nishimura et al.
(1318) proposed that countercurrent multiplication occurs by
502
single-solute recycling. They proposed that sodium chloride
transported out of the thick ascending limb without accompanying water (see above) is the single effect driving the
process, and that this sodium chloride is recycled by entering the thin descending limb, which has very high sodium
and chloride permeabilities and very low water permeability
(1318). Nishimura et al. (1318) further suggested that this process combined with a cascade of loops of increasing lengths
could establish the sodium chloride-based osmotic gradient
in the medullary cones. In the presence of AVT this osmotic
gradient would then cause water to move out of the parallel
collecting ducts, thereby concentrating the urine to the level
of the osmotic gradient (1318). As in the outer medulla of the
mammalian kidney, the thick ascending limbs would carry
fluid that is hypoosmotic to the plasma from the medullary
cones into the cortical region. An aquaporin 2 (AQP2) water
channel, which is a homologue of mammalian antidiuretic
hormone-responsive AQP2, is present in the apical membrane and subapical cytoplasmic region of quail collecting
duct cells, and it appears to increase in the apical membrane in
the presence of AVT (2053). However, the osmotic water permeability of quail collecting ducts is low and only increases a
modest amount in the presence of AVT (1319). Nevertheless,
a mathematical simulation of this proposed model (using the
anatomical and transport characteristics of quail nephrons)
generated a maximum U/P osmolality ratio of about 2.26,
a value consistent with what is actually observed in birds
(e.g., Table 18) (1021). This mathematical model also indicated that active sodium chloride transport out of the prebend
region of the thick ascending limb is of great significance in
producing the osmotic gradient. Moreover, further mathematical modeling (1020) indicated that the effectiveness of the
avian concentrating mechanism depends in part on the structural arrangement of tubules and vessels within the medullary
cones (a ring of collecting ducts surrounding a core of thin
limbs and capillaries, with thick limbs distributed inside and
at the periphery of that core) described by Nishimura et al.
(1318). The limited concentrating ability of the avian kidney
is significant with regard to urate excretion (see below) and
water reabsorption in the lower intestinal tract (see below)
and does not reflect the overall ability of birds to conserve
water.
Volume 4, April 2014
Comprehensive Physiology
Table 19
Osmoregulation and Excretion
Examples of Approximate Percent Total Urinary Nitrogen as Urates, Urea, and Ammonia
Percent urinary nitrogen as:
Urates
Urea
Ammonia
References
Chicken
Gallus gallus domesticus (O)
55-72
2-11
11-21
(1212)
Duck
Anas platyrhynchos (O)
54
1
29
(1762)
Anna’s
Hummingbird
Calypte anna (N)
35-65
8-13
27-53
(1480)
Turkey Vulture
Cathartes aura (C)
76-87
4
9-16
(1213)
O, omnivore; N, nectivores; C, carnivore.
Excretory End Products of Nitrogen Metabolism:
Ammonia, Urea and Uric Acid
Birds are generally considered to be uricotelic—that is, urate
is the primary form in which excess nitrogen is excreted (Table
19). However, a significant fraction (average about 20%) of
urinary nitrogen is in the form of ammonia in the urine of
most birds, and at least one nectivorous species (Anna’s humming bird, Calypte anna) can excrete over 50% as ammonium
salts when its intake of nectar is high at low temperatures
(Table 19) (1480). Thus, this species and perhaps some other
nectivores appear to be able to change from being uricotelic
to ammoniotelic (i.e., they are facultative ammoniouricoteles) (Table 19). In all avian species, urea accounts for only
a very small percentage of the nitrogen excreted in the urine
(Table 19).
Ammonia. Because ammonia is highly toxic, very little
is found in the blood and, therefore, almost none is filtered.
Instead, as in other vertebrates, it is produced and secreted by
the renal tubules themselves. Although ammonia appears to
play a significant role in nitrogen excretion in birds (and the
major role in a few species), its production and excretion are
also likely to be very important in maintaining acid-base balance by removing acid and generating an equivalent amount of
bicarbonate. Indeed, the production and excretion of ammonia have been shown to increase with an acid load in chickens
(Gallus gallus domesticus) (346, 1093).
Although the exact tubule site of ammonia production in
birds is unknown, the appropriate enzymes for its production
are found in homogenates of the whole chicken kidney and
in tubules separated from superficial regions of the kidney,
apparently from reptilian-type nephrons (346, 347). The activities of glutaminase I, glutamate dehydrogenase, and alanine
aminotransferase increase in the kidneys of acidotic chickens
(346). In addition, Craan et al. (346) found that the production
of ammonia is increased in superficial tubules from acidotic
chickens compared with those from control chickens when
Volume 4, April 2014
they are incubated in vitro with either alanine or glutamine.
However, because only the uptake of glutamine, not alanine,
is enhanced in tubules isolated from acidotic animals, Craan
et al. (347) suggested that glutamine is the preferred substrate
for enhanced ammonia production.
Neither the tubule site nor the mechanism of ammonia
secretion is known. However, as discussed above with regard
to reptiles, secretion could involve both non-ionic diffusion of
free ammonia (NH3 ) with trapping as ammonium (NH4 + ) in
the lumen and carrier-mediated transport of ammonium ions
(NH4 + ), depending on the pH of the luminal fluid and the
quantity of ammonia to be secreted. Because micropuncture
studies on starlings have indicated that the pH of the luminal
fluid is about 7.6 in the proximal tubules and about 6.4 in early
cortical collecting ducts (1013), it seems likely that carriermediated transport of ammonium ions would be most important in the proximal tubule where much of the ammonium is
likely to be produced, whereas non-ionic diffusion could play
a significant role in the collecting ducts. In any case, ammonia
is actually excreted in the urine as ammonium salts, probably either ammonium chloride or, in these uricotelic animals,
ammonium urate. It should be added that although ureteral
urine may enter the colon before it is excreted (see below),
recent studies have shown that the colonic epithelium has an
extremely low permeability for NH3 and is also capable of
secreting NH4 + into the colon lumen mediated by a vacuolar
type H+ -ATPase (794). Thus high ammonia concentrations
can be maintained.
Urate. Most uric acid is produced in the liver, exists as
urate salts in the plasma, is filtered by the glomerulus and is
secreted by the renal tubules. Although urates appear to be
freely filtered by the avian glomeruli, there is some evidence
that a small amount is bound to plasma proteins (387). In addition, variable amounts of urate can be synthesized by the renal
tubules. In fasted chickens, such urate apparently contributes
about 3% of that excreted, in normally fed chickens about
503
Osmoregulation and Excretion
Comprehensive Physiology
20% and in chickens systemically infused with hypoxanthine
about 50% (284).
Although urate is filtered, net secretion by the proximal
tubules contributes most of the urate in the avian urine (final
excretion of about five times the amount filtered) (1016).
Despite some urate production in the renal tubule cells, most
of this secreted urate involves transepithelial transport from
the peritubular blood to the tubule lumen. In vivo micropuncture studies on superficial loopless reptilian-type nephrons in
starlings and in vitro microperfusion studies on transitional
looped nephrons in chickens clearly show that net secretion occurs along the proximal tubules (205, 1016). Studies
with isolated, perfused and non-perfused chicken proximal
tubules and primary cell cultures indicate that the net secretion involves uptake into the cells against an electrochemical
gradient and movement from the cells into the lumen down an
electrochemical gradient (205, 455). Inhibitory studies suggest that much, but not all, of the urate transport into the cells at
the basolateral membrane involves the same pathway as paraaminohippurate (PAH) and other organic anions and involves
countertransport for α-ketoglutarate (αKG) (Fig. 32). However, at least a portion appears to involve some other pathway
with probable countertransport for another unknown anion
(Fig. 32). In mammals, the basolateral exchange of organic
Lumen
Cell
–2mV
Peritubular fluid
–70mV
0mV
Urate–
? OAT ortholog
?A–
Urate–
Urate– or PAH–
?
MRP4
ortholog
?
?
? OAT ortholog
PAH
αKG2–
αKG2–
Metabolism
?
Na+
Na+
K+
K+
ATP
ADP
Urate–
Figure 32
?
Model for net tubular secretion of urate and PAH in avian
proximal tubules, based on studies in chickens. Filled circle with solid
arrow indicates primary active transport. Open circle with solid arrow
indicates secondary active transport. Open circle with broken arrow
indicates carrier-mediated passive transport. Therefore, for countertransport, solid arrow indicates movement against electrochemical gradient; broken arrow indicates movement down electrochemical gradient. A− indicates anion of unspecified nature. Question marks indicate
that these processes have not been clearly demonstrated.
504
anions (e.g., PAH) for αKG involves the organic anion transporters (OATs). Since mRNA for OAT-like organic anion
transporters has been found in chicken proximal tubule epithelium (455), it seems likely that OAT orthologs are involved
in the basolateral transport of urate and other organic anions
(e.g., PAH) in avian proximal tubules (Fig. 32). There is some
unilateral backflux of urate from lumen to peritubular fluid,
and because no urate accumulates in the tubule cells during
this process, it seems likely that this backflux moves between
the cells (Fig. 32) (205).
Although the transport step for both urate and PAH from
the tubule cells to the lumen in birds is down an electrochemical gradient, little is known about it. However, mRNA for
a multidrug resistance peptide 4 (MRP4)-like transporter is
found in chicken tubule epithelium, and inhibitor studies suggest that it may be involved in the process (Fig. 32) (455).
If this is the case, of course, then this luminal flux would
not be completely passive because this is an ATP-dependent
transporter.
Although the concentration of urate in avian plasma is
well within the solubility range of urate salts (see section on
Reptiles for solubilities), the concentrations developed in the
lumens of the renal tubules exceed even that for potassium
urate and are well above those for sodium urate or ammonium urate (1016). Although there is visual evidence for an
apparent urate precipitate in the collecting ducts of starlings
(1016), any precipitation in the proximal and distal tubules
of avian nephrons is prevented by binding to serum albumen,
which, as noted above, is filtered to a significant extent in
birds (183, 259, 1758). Urate and its associated albumen form
small spheres (0.5-0.14 mm in diameter) (190) beginning in
the early part of the proximal tubule (245, 260). Because
of the large amounts of protein, these spheres are held in a
colloidal suspension in the tubule lumen (190) so that precipitation only begins to occur in the more concentrated urine of
the collecting ducts (1016). However, the limited urine concentrating ability of the avian kidney (see above) probably
prevents much greater precipitation before the urine enters
the lower gastrointestinal tract. As noted above in the discussion on reptiles, the inorganic cations of the urate salts
trapped in colloid suspensions or in these precipitates would
not contribute to the osmolality of the aqueous phase of the
ureteral urine, and therefore the limited concentrating ability
of the kidney does not accurately reflect its ability to excrete
inorganic cations in the ureteral urine.
The large amount of protein associated with urate is actually reclaimed in the lower gastrointestinal tract (see below).
In this region, bacteria break down the protein, allowing the
resulting peptides and amino acids to be reabsorbed by the
colonic epithelium (245, 260), and no protein appears in
the urine that is finally excreted (841). Finally, about 60% of
the uric acid entering the colon is also broken down, permitting reabsorption and reutilization of nitrogen, if necessary, as
may be the case in nectivorous and frugivorous species (30,
183, 883).
Volume 4, April 2014
Comprehensive Physiology
Lower Gastrointestinal Tract
Substantial modification of the ureteral urine can occur in the
lower gastrointestinal tract (coprodeum and colon) and this
has been extensively studied, particularly by Skadhauge and
his colleagues (e.g., see the following reviews [620, 1017,
1018]). Most of the studies were undertaken on domestic
fowl (Gallus gallus domesticus), but similar results have been
observed in other species. Only a few aspects are mentioned
in this review. The ureteral urine first enters the urodeum
and then moves retrograde driven by peristaltic movements
first into the coprodeum and then into the colon (and cecae
in those species in which they are found) (183). The extent
of this retrograde peristalsis from the coprodeum into the
colon appears to be regulated in part by the sodium concentration and osmolality of the ureteral urine, being marked
at low osmolalities and low sodium concentrations but tending to cease when the osmolality is more than 100 mOsm
above the osmolality of the plasma (211). This is significant
with regard to solute-linked water reabsorption in the colon
(see below).
Both the coprodium and colon are capable of reabsorbing sodium chloride, but this process is influenced by sodium
chloride intake and aldosterone. Moreover, there are distinct
differences in the transport processes between the coprodeum
and the colon. Most of the studies on the sodium transport
process have been performed in vitro where, under voltage
clamp conditions, the short-circuit currents (ISC ) are virtually
identical to active sodium reabsorption, as determined by isotopic flux measurements (292, 1065, 1066), but comparable
data are observed in vivo.
Coprodeum. When birds are maintained on a high sodium
chloride diet or a medium sodium diet (such as that found
in commercial feed), sodium reabsorption and ISC in this
region are essentially zero. However, when the animals are
adapted to a low sodium chloride diet, sodium is reabsorbed
at a very high rate (net sodium flux: 14 μEq cm−2 ·hr−1 ;
ISC : 380 μA·cm−2 ) (51, 292, 304). During this high-sodium
transport under open-circuit conditions in vitro and in
vivo, a lumen-negative transepithelial potential difference of
40-60 mV develops and drives passive chloride reabsorption
to accompany the sodium. This dramatic change from almost
no sodium reabsorption during adaptation to a high-sodium
diet to a very high rate of sodium reabsorption during adaptation to a low-sodium diet results from activation of amiloridesensitive sodium channels (ENaCs) in the luminal membrane
(134, 296, 305). The energy-requiring sodium transport step
at the basolateral membrane involves Na+ -K+ -ATPase, which
does not change with changes in sodium chloride in the diet
(1190). Stimulation of sodium transport similar to that produced during a low-sodium diet can be produced by aldosterone, the plasma level of which is maximal with adaptation
to a low-sodium diet and nearly undetectable with adaptation to a high-sodium diet (51, 303, 304, 1093, 1700). Finally,
there is no absorption of glucose or amino acids and no solutelinked water flow in the coprodeum (135, 1539).
Volume 4, April 2014
Osmoregulation and Excretion
Colon. Sodium reabsorption in the colon, as in the
coprodeum, changes with adaptation to different sodium
diets. However, the processes involved are substantially more
complex. During adaptation to a low-sodium diet, sodium
reabsorption, as in the coprodeum, involves an electrogenic,
ENaC-mediated process of similar magnitude. Also, as in the
coprodeum, there is no stimulation of sodium transport by
amino acids or hexoses. However, the epithelium of the colon
is leakier than that of the coprodeum (resistance of about 80100 ohm·cm2 versus 300-500 ohm·cm2 ) and, therefore, has a
lower transepithelial PD (15-20 mV versus 40-60 mV). Along
with this, there is substantial solute-linked water reabsorption
that can occur against an osmotic pressure gradient of 100180 mosmol/kg (lumen > blood) (137, 618, 619, 1539, 1705).
Therefore, as long as ureteral urine does not have an osmolality more than about 100-180 mosmol/kg greater than the
plasma, it will move retrograde (see above) into the colon, and
the solute-linked water reabsorption will offset movement of
water into the hyperosmotic lumen. More concentrated urine,
which at most is only about 300 mosmol/kg above the plasma
osmolality, will remain in the coprodium before excretion.
With adaptation to a high-sodium diet, ENaC channel
activity in the luminal membrane almost completely disappears, as it does in the coprodeum. However, in the colon,
sodium reabsorption continues with the entry step across the
luminal membrane involving a number of other processes.
The most important of these are apparently sodium-coupled
glucose and amino acid cotransport (51, 303, 1065, 1066,
1538). Sodium-glucose cotransport is attributed to one of the
sodium glucose luminal transporters (SGLTs) (136, 1014).
Thus, most of this continued sodium transport is dependent
on the presence of glucose and amino acids in the lumen.
At the same time, these processes help reclaim glucose and
amino acids still present in the ureteral urine and in the chyme
moving down the gastrointestinal tract.
In addition to entry via these cotransporters, sodium
can cross the luminal membrane via sodium-hydrogen
countertransport, apparently involving the sodium-hydrogen
exchanger 2 (NHE2) isoform (403, 444). Overall, the colon
has a much higher total transport capacity than the coprodeum.
This suggests that the colon does much of the work of
sodium and water reabsorption from ureteral urine and chyme,
whereas the coprodeum functions to fine tune the sodium
excretion (1821).
Caeca. Not all bird species have caeca. However, in vivo
and in vitro studies of transport in the main body of caeca of
domestic fowl have revealed substantial amiloride-sensitive
reabsorption of sodium (670, 671, 1538, 1823). Moreover,
high rates of solute-coupled water reabsorption accompany
the sodium reabsorption, indicating that the caeca may play
an important role in overall solute and water balance (1538,
1822, 1823). As in the coprodeum and colon, the reabsorption
of sodium is enhanced by adaptation to a low sodium diet and
by aldosterone (671, 1823). Dehydration can also enhance net
sodium reabsorption and its accompanying water flux (1538,
505
Osmoregulation and Excretion
1823). Taken together, these data indicate that the caeca, along
with the colon, may play an important role in overall solute
and water balance in those species that have them.
Comprehensive Physiology
Blood
Na
Lumen
2Cl
Salt Glands
As noted above, the limited urine-concentrating ability of
birds is closely related to the need to excrete urate, itself
a water-conserving process, and the solute coupled water
absorption in the lower intestine. However, in marine birds
and birds that can adapt to a marine habitat, the need to
excrete sodium chloride and generate free water far exceeds
the capabilities of their kidneys. This requirement is met by
secretion of a highly concentrated sodium chloride solution by
paired supraorbital nasal salt glands, as first described by Knut
Schmidt-Nielsen and his colleagues (1631). All birds examined have at least rudimentary salt glands (1159), but their
development and functional activity depend on the amount of
sodium chloride consumed by the animals (1684).
The gross and fine structure of these glands, as well as
their blood supply and innervation, have been well described
and reviewed by a number of investigators (232, 602, 803) and
will not be discussed in detail here. However, as noted with
regard to reptiles (see above), the glands consist of secretory
tubules. Groups of these tubules form lobules in which all of
the tubules empty into a common central ductule. A number
of these ductules empty into primary ducts, which in turn
empty into two main ducts, one for each gland. These ducts
carry the secreted fluid to the nasal cavity (232) where it is
removed by passive dripping or shaking of the head.
As also noted in the section on reptiles (see above) the
epithelium of each of the secretory tubules has poorly differentiated, apparently non-secretory cells near the blind end.
The cells then become progressively more differentiated along
the length of the tubules, developing extensive infoldings
of the basolateral membrane, along which Na+ -K+ -ATPase
is localized (151, 499, 797), and an increasing density of
mitochondria, as expected of ion-transporting epithelium. A
hyperosmotic sodium chloride solution, with only minor contributions from other ions, is secreted by these cells in all
birds studied when they are given an osmotic stimulus. The
process by which the initial secretion is considered to occur
involves secondary active chloride transport and is illustrated
in Fig. 33. Chloride enters the secretory cells across the basolateral membrane via the Na+ -2Cl− -K+ cotransporter (500,
1843) and exits across the luminal membrane via chloride
channels; potassium is returned to the basolateral fluid via
basolateral potassium channels (1175, 1541). Sodium accompanies the chloride via a paracellular pathway (Fig. 33). The
active, energy-requiring step, which drives the whole process,
involves the basolateral Na+ -K+ -ATPase, which maintains a
low intracellular sodium concentration. As in the case of the
reptilian salt glands (see above), the process by which the
final sodium chloride concentration in the secreted fluid is produced is unclear. Shuttleworth and Hildebrandt (1684) suggest
506
Ca2+
K
K
Cl
Na
Na
K
Na
Figure 33 Diagram illustrating the proposed mechanism of secretion in the avian salt gland. Increases in cytosolic Ca2+ activate secretion by increasing the opening of basolateral Ca2+ -activated K+ channels and apical Ca2+ -activated Cl− channels. Reproduced from Shuttleworth and Hildebrandt (1684) with permission.
that the most likely mechanism involves hyperosmotic secretion by the cells with no further modification along the ducts.
In any case, the concentration of sodium chloride in this solution can reach as much as 10 times that in the plasma, in some
species (1411). Moreover, Kaul et al. (904) reported that the
glands in saline-adapted ducks are capable of removing more
than 20% of the sodium in the blood passing through them.
This sodium chloride secretory process in the salt glands of
reptiles and birds is essentially the same as that in the rectal
glands of elasmobranchs and the gills of marine teleosts (see
section on Agnatha and Pisces and Fig. 18).
Regulation of the avian salt glands involves (1) progressive adaptation upon exposure to a salt load and (2) rapid
activation of the secretory process in the already salt-adapted
gland. Control certainly appears to involve central osmoreceptors as originally suggested by Schmidt-Nielsen (1631),
but extra-cerebral osmoreceptors may play some role (697).
In any case, the output from these receptors is conveyed to
the salt glands via cholinergic fibers in the secretory nerve
that stimulates each gland (571, 697). Parasympathetic fibers
in this nerve have endings that carry both vesicles filled with
acetylcholine (796) and vesicles filled with vasoactive intestinal peptide (601, 1104), which are released upon electrical stimulation. However, as reviewed by Shuttleworth and
Hildebrandt (1684), regulation of both of the responses noted
above involves neuronal release of acetylcholine, which interacts with muscarinic receptors on the secretory cells. This
leads, in turn, via the phosphatidylinositol system, to an
increase in the free calcium concentration in the cytoplasm.
The way in which this one signaling pathway leads to the two
separate responses has been studied by Shuttleworth and his
colleagues and is reviewed in Shuttleworth and Hildebrandt
(1684). This review should be consulted for further details.
In any case, the nasal salt glands are sufficiently effective to
permit some species of birds to thrive in areas where little
freshwater is available.
Volume 4, April 2014
Comprehensive Physiology
Nitrogen Excretion
Origin of Nitrogeneous Compounds
Ammonia
Ammonia (i.e., the total of NH3 and NH4 + , TAmm ) is present in
terrestrial and aquatic environments. It is generated by atmospheric fixation due the breakdown of N2 by lightning, by
decomposition of nitrogenous organic material and by energetically costly nitrogen fixation of microorganisms utilizing the oxygen-sensitive nitrogenase enzyme system (805,
1488). Large amounts of nitrogenous material are produced
by anthropogenic activities and a significant proportion of this
is eventually converted into ammonia. Anthropogenic ammonia derives primarily from the production and use of fertilizers, biomass burning and from intensive animal husbandry
(15, 839).
In addition, animals produce and excrete nitrogenous
molecules. The vast majority of these nitrogenous molecules
are the three main waste products, namely ammonia, urea and
uric acid. Synthesis and pathways of these metabolic endproducts in animals have been extensively described by J. W.
Campbell (248) and will therefore be covered only to a minor
extent in this article. Most of the nitrogen of the major nitrogenous waste products derives from the degradation of amino
acids, in order to utilize the remaining carbohydrate skeleton
for energy production or storage as glycogen or triglycerides
(968). The main pathway for amino acid catabolism requires
an initial transfer of the α-amino group of a given amino
acid to α-ketoglutarate by an aminotransferase (also known
as transaminase) to form glutamic acid (glutamate) and the
corresponding α-keto acid. Glutamate is then transported into
the mitochondria, where the amino acid is oxidatively deaminated by glutamate dehydrogenase (193).
Serine and threonine can be deaminated directly by
serine-dehydratase and threonine-dehydratase, respectively.
Serine-dehydratase is present in mammalian hepatocytes,
here almost exclusively in the cytoslic compartment (1787).
Serine-dehydratase was also found in invertebrates, for example, in the gills, the digestive diverticula and mantle and
foot muscle of the brackish-water bivalve Corbicula japonica (716). However, due to its low activity in invertebrates
other than molluscs, the role of serine-hydratase in amino
acid catabolism and ammonia generation in invertebrates is
unclear.
Proline is often used directly as an energy source in invertebrates, for example, in tissues with high oxidative rates such
as insect flight muscles (63, 1593) or in the mantle tissues of
cephalopods (1782). After the initial conversion of proline to
-pyrroline-5-carboxylate, the product is then further converted into glutamate (777).
A second pathway of amino acid metabolism is the socalled purine nucleotide cycle. This system plays supposedly only a minor role in some specific mammalian tissues
(1102, 1103). In invertebrates, however, this system might
be of more importance. In this mechanism, transaminase
Volume 4, April 2014
Osmoregulation and Excretion
reactions are coupled with the purine nucleotide cycle, which
is involved in the synthesis of nucleic acids. Here inosine
monophosphate (IMP) and aspartate, which is synthesized
from oxalacetate and the α-amino group derived from the
deamination of glutamate, form adenylosuccinate, a precursor of adenosine monophosphate (AMP). After deamination
of AMP by AMP deaminase, ammonia is released and IMP
enters the cycle again. As reviewed by Campbell, bivalve and
gastropod molluscs have high aspartate animotransferase and
AMP deaminase activities but low glutamate dehydrogenase
activity (248). Also, adenylosuccinate synthase and adenylosuccinate lyase were found in the hepatopancreas of the
gastropod Helix aspersa (249), indicating the putative participation of the purine nucleotide cycle in ammonia generation
in molluscs.
As in other animals, the vast majority of the excreted
ammonia in crustaceans originates from the catabolism of
proteins and amino acids. Some additional ammonia is produced in reactions involving purine and pyrimidine bases
(307, 1950). Ammonia derived via this uricolytic pathway
is considered to contribute only a small portion of the total
excreted compared to the predominant production from amino
acids (705, 1634). Ammonia derives for the most part from
deamination of glutamine, glutamate, serine and asparagine
by the specific enzymes glutaminase, glutamate dehydrogenase, serine dehydrogenase and asparaginase, respectively
(643, 926, 969). The purification and characterization of AMP
deaminase from crustaceans (1752) raised the possibility that
ammonia could also be produced by deamination of adenylate
via the purine-nucleotide-cycle. Further, confirmed by analyzing expressed sequence tags (ESTs), mRNA coding for adenylosuccinate synthase is indeed expressed in tissues of lobster
Homarus americanus (Genbank accession # EH116452) and
the green shore crab Carinus maenas (Genbank accession
# DN550904). The role of this particular pathway, however, is
not clear to date. The amount of metabolic ammonia produced
in crab gill epithelium itself is significant. In marine stenohaline Cancer pagurus crabs, the excretion due to branchial
metabolic ammonia was 12.3 μmol gFW−1 h−1 . About 50%
of these rates were found in gills of the osmoregulating crabs
Carcinus maenas and Eriocheir sinensis (1945). For comparison, the overall excretion rates of total ammonia in Cancer
pagurus, Carcinus maenas and Eriocheir sinensis were only
ca. 340, 140 and 120 nmol gFW−1 h−1 , respectively (1945).
Urea
Urea is the primary waste product of adult amphibians, turtles, mammals and some invertebrates. Here toxic ammonia, generated in the mitochondria, is converted into urea
for excretion. Besides being a less toxic nitrogenous waste
product compared to ammonia, urea is also synthesized
for osmoregulatory purposes. While in mammals urea is
used in the kidneys to produce a hypotonic urine (385), it
serves in other vertebrates as an osmolyte to balance osmotic
507
Osmoregulation and Excretion
gradients between the body fluids and the environment (926).
The fully aquatic and ammonotelic African claw frog Xenopus
laevis becomes ureotelic when acclimated to 400
mosmol·kg−1 solutions of NaCl (2031). In marine elasmobranchs, urea is an important osmolyte with blood
concentrations of 300 to 400 mmol/l. Urea is synthesized in the ornithine-urea-cycle (OUC), which has been
reviewed extensively by Meijer and others (1217). A
key enzyme in this process is the carbamyl phosphate
synthetase (CPS). In animal systems, three CPSs exist:
CPS-I, CPS-II and CPS-III. In ureotelic mammals and
amphibians, CPS-I utilizes ammonia as a substrate and
N-acetyl-l-glutamate for activation. CPS-I functions in hepatic ammonia detoxification (1498). CPS-II does not require
N-acetyl-l-glutamate and utilizes glutamine as a substrate
(639). CPS-III is involved in pyrimidine synthesis. Similar
to CPS-II, the mitochondrial enzyme CPS-III utilizes glutamine as a substrate, but in contrast to CPS-II, it requires
N-acetylglutamate. CPS-III is involved in urea synthesis in
invertebrates and elasmobranchs, were urea is used as osmotic
ballast (32) (258, 1852, 1853). For a more detailed review on
the ornithine-urea-cycle and its regulation, see also Campbell (248, 1986). In most invertebrates and teleost fish, urea
is synthesized by uricolysis or argininolysis (621, 2040). In
general, teleost fish are ammonotelic, and enzymes necessary
for a functional urea cycle are suppressed (812). However,
some teleost species synthesize fair amounts of urea and can
become ureotelic. This can occur in response to environmental conditions that limit ammonia excretion, for example, in
the tilapia Oreochromis alcalicus grahami (1509) living in
highly alkaline lakes (pH ≈ 9.6-10) or the air-breathing Asian
stinging catfish Heteropneustes fossilis (1594). For the Gulf
toadfish Opsanus beta it has been speculated that urea production and excretion is used as a protective mechanism to
cloak the scent of ammonia that might lure predators (73,
1202). All these species express the full complement of OUC
enzymes. In contrast, insects lack one or more genes-encoding
enzymes required for a fully functional OUC (2069). Scaraffia and others discovered that in addition to urea production
by arginase-catalyzed hydrolysis of dietary arginine, the yellow fewer mosquito Aedes aegypti synthesizes urea in an
amphibian-like uricotylic pathway (1617).
Uric acid
In arachnids, reptiles, birds and crocodilians, excessive
ammonia from amino acid catabolism is mostly converted
into uric acid or other purines for excretion. Uric acid is
relatively non-toxic and very little water is required for its
excretion. Using uric acid as the primary nitrogenous waste
product is therefore a major water conservation mechanisms.
However, its synthesis is energetically very expensive. Uric
acid and other purines are also formed for excretion by
land-living snails and arthropods, including arachnids, insects
and the terrestrial robber crab Birgus latro (645). To date it
is not clear whether uric acid synthesis in invertebrates is
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Comprehensive Physiology
analogous to the pathways found in the liver of higher vertebrates. Pathways for uric acid synthesis and its regulation
have been reviewed in detail by Campbell (248).
Toxicity of Ammonia
Ammonia is highly toxic in animals and at higher levels also
in plants (198). With a pK of 9.2 to 9.8, which depends on
the temperature and salinity of the media (243), ammonia is
a weak base. In body fluids with a physiological pH ranging roughly from 7.2 to 7.8, approximately 95% to 99%
of total ammonia occurs in the hydrated form NH4 + . Both
forms of ammonia, NH3 and NH4 + , have toxic effects by
potentially disturbing the cytosolic and/or intraorganelle pH.
For instance, NH3 sequestered by acidic organelles due to
ammonia trapping (see below) can impair proper functioning of Golgi vesicles and lysosomal proteases by shifting
the intraorganelle pH away from its optimum necessary for
normal operation. Ammonia formed from glutamate deamination is generated within the mitochondria, where its toxicity
results from disruption of the proton gradient across the inner
mitochondrial membrane. Due to its relative alkalinity (pH
mitochondria vs. cytoplasm ≈ 0.8), an outwardly directed
PNH3 is given. Along this gradient NH3 exits the mitochondrial matrix and binds to protons in the intermembrane space,
thereby abolishing the pH gradient, which drives oxidative
phosphorylation. Ammonia thereby acts as an H+ -gradientuncoupler (1338).
Hydrated NH4 + and K+ ions have the same ionic radius
of 1.45 Å (950, 1957) and due to their K+ -like behavior,
ammonium ions affect the membrane potential, for example,
in the giant axon of Loligo pealei (139) and in mammalian
neurons (337). Ammonia also exhibits an inhibitory effect on
epithelial Na+ channels (ENaC) when expressed in Xenopus
oocytes (1293). The mode of inhibition is not fully understood, however.
In mammals, elevated ammonia causes major damage in
the central nervous system, including changes in blood-brain
barrier morphology (1012). In addition, elevated ammonia
levels in mammals have been related to Alzheimer disease
(Alzheimer Type II astrocytosis) due to toxic accumulation
of glutamine in astrocytes, which leads to cell swelling and
cell death (236). Interestingly, in the ammonia-tolerant Gulf
toadfish Opsanus beta, the hydration status of the brain does
not change with increased brain glutamine levels in ammoniastressed animals (1906, 1929).
In microglia and astroglioma cell lines, ammonia affects
major functional activities such as phagocytosis and endocytosis. In addition, ammonia modifies the release of cytokines
and increases the activity of lysosomal hydrolases (52,
53). Ammonium ions inhibit important enzymes involved
in metabolism, such as isocitrate dehydrogenase and αketoglutarate dehydrogenase (337).
Marcaida and coworkers found evidence that ammonia
toxicity is mediated by excessive activation of N-methyl-Daspartate (NMDA)-type glutamate receptors in the brain.
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Comprehensive Physiology
As a consequence, cerebral ATP depletes while intracellular Ca2+ increases, with subsequent increases in extracellular K+ and finally cell death (1154). In addition
to this, neurotoxicity is mediated by a direct inhibitory
effect of ammonia on the astrocytic EAAT-1 (GLAST) and
EAAT-2 (GLT-1) transporters, which are responsible for the
removal of glutamate from the neuronal synapse (270, 948,
1326).
In crustaceans, for example in the lobster Homarus
americanus (2062) and the crayfish Pacifastacus leniusculus (700), elevated ammonia levels in low-salinity media disrupt ionoregulatory function. Exposure of the green shore
crab Carcinus maenas to 1 mmol l−1 total ammonia leads to
increased ion permeability and salt flux across the gill; higher
concentrations reduce both variables (1740). In Penaeus
stylirostris, elevated ammonia levels reduced the total number
of immune active haemocytes by about 50% (1022). In the
Dungenees crab Metacarcinus magister, a two-week exposure
to 1 mmol l−1 total ammonia causes an increase of hemolymph
ammonia to environmental levels, paralleled with a total loss
of the branchial capability to actively excrete ammonia and
a decrease in branchial mRNA expression levels of a Rhlike ammonia transporter, V-ATPase, Na+ /K+ -ATPase and a
cation/H+ exchanger, all transporters suggested to be involved
in ammonia transport processes (1174, 1953). Most literature
dealing with ammonia toxicity in non-mammalian systems
can be found for fish. Several review articles on this subject
have been recently published, such as by Eddy (472), Ip and
others (831, 832) and Randall and Tsui (1507, 1874). Among
other effects, it was found that increased water ammonia levels decreased critical swimming velocity in rainbow trout and
coho salmon (1210, 1667, 1986) and impaired the performance of the “fast-start escape response” in the golden gray
mullet Liza aurata L. (1209). Further, branchial gas exchange
and oxidative metabolism are disturbed by excess ammonia
(1997). Based on the studies by Alabaster and Lloyd (15)
and on data gained on Atlantic salmon (Salmo salar) (16),
it was speculated that a reduction in the level of dissolved
oxygen in the water increases the toxicity of ammonia to
fish (472).
Due to its toxicity, in most species, including mammals (337), fish (666) and aquatic crabs (242, 1945), the
ammonia concentration of the body fluids is typically low
(50-400 μmol. l−1 ). Concentrations exceeding 1 mmol. l−1
total ammonia (NH3 + NH4 + ) are usually toxic to mammalian cells (808). In aquatic crustaceans, environmental
exposure to ammonia is lethal at relatively low doses. For
instance, LC50 after 96 hours of exposure was determined
in the crayfish Orconectes nais at 186 μmol. l−1 NH3 (720),
in the Sao Paulo shrimp Penaeus paulensis at 19 μmol. l−1
NH3 and 0.307 mmol. l−1 total ammonia (1386) and in the
redtail prawn Penaeus penicillatus 58 μmol. l−1 NH3 and
1.39 mmol. l−1 total ammonia (279). In contrast, terrestrial
crustaceans such as Porcellio scaber (2036) or Cardisoma
carnifex (2022) exhibit a much higher tolerance to elevated
hemolymph ammonia levels.
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Osmoregulation and Excretion
Nitrogen Transporters
Ammonia transporting proteins and mechanisms
Ammonia occurs in a pH-dependent equilibrium either as
gaseous, uncharged NH3 or in its ionic form NH4 + . Due to
it relatively high pK (9.2-9.8), the vast amount of ammonia exists in physiological solutions as NH4 + , although small
amounts of NH3 will always be present. It has long been
thought that transepithelial ammonia transport occurs mainly
by membrane diffusion of uncharged NH3 driven only by
an existing or generated partial pressure gradient (PNH3 ). An
excretion mechanism solely based on NH3 membrane diffusion is not likely, however, because membrane permeability
of NH3 is much lower than that of CO2 (950). Indeed, some
plasma membranes of animal epithelia are relatively impermeable to NH3 as shown for frog oocytes (221), the renal
proximal straight tubules (591) and mammalian colonic crypt
cells (1697). Therefore, in addition to limited NH3 membrane
diffusion and NH4 + crossing epithelia via paracellular routes,
for transmembrane movements of ammonia transport proteins
are required.
Na+ /K+ -ATPase Perhaps the most important transporter
in ammonia-excreting epithelia is the basolateral Na+ /K+ ATPase, which transports NH4 + actively from the body fluids into the cytoplasma of epithelia cells. Soon after Jens
Skou discovered the “K-pump” in leg nerves of the green
shore crab Carcinus maenas, he demonstrated that this pump
also accepts NH4 + as a substrate, replacing K+ ions (1708)
(Fig. 34A-1).
Masui et al. (1181) showed that the branchial Na+ /K+ ATPase from the crab Callinectes danae is synergistically
stimulated by NH4 + and K+ , increasing its catalytic activity by up to 90%. The authors came to the conclusion that
the two ions bind to different sites of the branchial Na+ /K+ ATPase. This observation was also attributed to the branchial
Na+ /K+ -ATPase of the freshwater shrimp Macobrachium
olfersii by Furriel et al. (569), who suggested that for this
species, at high NH4 + concentrations the pump exposes a
new binding site for NH4 + that, after binding to NH4 + , modulates the activity of the Na+ /K+ -ATPase independently of
K+ ions. In osmoregulating blue swimmer crabs Portunus
pelagicus, ammonia activates branchial Na+ /K+ -ATPase but
without interactive effects of NH4 + and K+ (1571). In the
gills of Carcinus maenas acclimated to brackish water, both
gradient-driven (1106) and active ammonia excretion (1946)
are partially inhibited by ouabain. Further, Mangum and
others demonstrated that ouabain inhibits ammonia excretion in the lamellibranch mollusc Ragia cuneata and the
polychetous annelids Nereis succinea and Nereis virens by
∼40%, 30% and 60%, respectively (1149). Similar observations were made in the freshwater flatworm Schmidtea
mediterranea. Here ouabain reduced the excretion of total
ammonia by approximately 50% (1947). Also for vertebrates
it was shown that the Na+ /K+ -ATPase is directly involved
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Osmoregulation and Excretion
Comprehensive Physiology
in ammonia transport processes. In the seawater fish Opasus beta and Takifugu rubripes, branchial enzyme activity
was similar when K+ was replaced by NH4 + ions (813,
1147). Further, when exposed to high environmental ammonia (HEA) concentrations, ouabain inhibited active ammonia excretion in the mudskipper Periophthalmodon schlosseri
(1508). Studies in the mammalian kidney confirmed Na+ /K+ ATPase mediated transport of ammonia, for example in the
inner medullay collecting duct (1925), or proximal tubule
(590, 983).
H+ /K+ -ATPase Functional
heterologous expression
studies in Xenopus oocytes revealed that ammonia can also
be transported by the apical localized colonic H+ /K+ -ATPase
(cH+ /K+ -ATPase), with NH4 + substitute for K+ ions (133,
340, 1794) (Fig. 34A-5). In addition, a study by Swarts and
others showed that the enzyme is more active in the presence
of NH4 + ions than in the presence of K+ ions (1794). The
cH+ /K+ -ATPase is therefore potentially involved in active
ammonia transport to protect and counteract mucosal to
serosal ammonia influxes in the mammalian colon where
extremely high ammonia concentrations can be found due to
protein degradation by intestinal bacteria (1118).
NKCC Besides directly transporting ammonia, the
Na+ /K+ -ATPase generates a low intracellular Na+ concentration, which drives secondary active ammonia transports,
for instance by the Na+ -K+ -2Cl− cotransporter (NKCC) and
Na+ /H+ -exchanger (NHE). Using the inhibitor furosemide
Good and others demonstrated that the apical NKCC in the
mammalian medullary thick ascending limb (MTAL) recognizes NH4 + as a substrate (625) (Fig. 34A-3). Also, experiments employing rabbit membrane vesicles from MTAL confirmed that NH4 + is accepted by the binding K+ site of the
furosemide- and bumetanide-sensitive NKCC with an affinity
similar to that for K+ (927). In T84 cells, a human intestinal
carcinoma cell line, flux studies showed that the basolateral
localized NKCC1 mediates blood to cell ammonia uptake
(2032).
In fish, studies on the perfused dogfish pup (Squalus acanthias) suggest that a portion (∼17%) of branchial ammonia
excretion is mediated by the basolateral-situated bumetainidesensitive NKCC (511). Further, sustained elevated expression levels of branchial NKCC1 in the pufferfish T. rubripes
in response to elevated environmental ammonia indicate the
importance of the cotransporter in fish ammonia excretion.
Cation/H+ exchanger (NHE) (Fig. 34A-4) Also
(A)
Ammonia
trapping
(B)
K+/NH4+
ATP
Na+
Na+
1
2
NH4+
4
NH3
8
NH3
K+/NH4+
ATP
6
K+/NH4+
3
7
2 Cl–
Na+
H+
H+ + NH3
NH4+
NH4+
ATP
H+
Unstirred
boundary
layer
5
?PNH3
(C)
NH3
H+
ATP
NH4+
Vesicular
transport
Figure
34 Ammonia-transporting proteins and ammoniatransporting mechanisms. Transporters and putative mechanisms are
explained in the text. (A) Fictive cell carrying basolateral Na+ /K+ ATPase (1), apical or basolateral K+ channels (2), apical or basolateral
NKCC-cotransporter (3), apical or basolateral cation/H+ exchanger
(4), apical H+ /K+ -ATPase. (B) Ammonia-trapping mechanism is
energized by H+ -ATPase (6). The resulting PNH3 is driving NH3
membrane diffusion (7) or facilitated NH3 diffusion via Rh-proteins,
Amts or MEPs (8). (C) Vesicular ammonia transport based on ammonia
trapping and vesicular NH4 + transport to target membrane.
510
driven by the Na+ /K+ -ATPase are Na+ -dependent cation
exchanger (NHEs). Early studies on crustaceans suggested
the participation of apical localized cation/H+ exchangers
(NHEs) in ammonia excretory mechanisms. These suggestions were based on the inhibitory effects of amiloride, a rather
unspecific blocker for Na+ channels and at higher dosage
also for NHEs (89). Hunter and Kirschner (815) reported a
substantial reduction in ammonia excretion of approximately
32% and 56% in the marine osmoconforming crabs Cancer antenarius and Petrolisthes cinctipes, respectively, when
the exchanger is blocked. Gill perfusion experiments in the
green shore crab C. maenas (1106, 1946) and C. pagurus
(1945) showed similar results. However, results obtained by
using amiloride on epithelia or animals with a cuticle must
be reviewed with caution. Experiments employing the isolated cuticle from Carcinus maenas have shown that cuticular Na+ and NH4 + conductances (Gcut ) are inhibited by apically applied amiloride in a dose-dependent manner, with an
inhibitor constant Kami-Na+ = 0.6·μmol·l−1 for sodium ions
and Kami-NH4+ = 20.4·μmol·l−1 for ammonium ions, respectively (1375, 1954). Further evidence of the participation
of cation-proton exchangers in ammonia transport processes
were found in the midgut of the tobacco hornworm Manduca sexta. Here luminal-applied amiloride blocked the active
ammonia uptake in a dose-dependent manner, suggesting an
ammonia transport by a so far non-characterized cation/NH4 +
exchanger (144, 1943).
In mammals, NHE-3 has been implicated to play a role
in renal ammonia transport (1270, 1957). Accordingly it
was demonstrated that proximal tubule brush border vesicles
exhibit Na+ / NH4 + exchange activity (928) (Fig. 34A-4).
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Comprehensive Physiology
Osmoregulation and Excretion
K+ channels Potassium ion channels are putative can+
+
didates to mediate transmembrane NH4 transport. K and
NH4 + ions have nearly identical biophysical characteristics
(1957) and NH4 + potentially substitutes the potassium ion at
the K+ -binding site of K+ -channels (Fig. 34A-2). As summarized by Choe and others, transport of NH4 + by K+ -channels
has been shown for various K+ -channel families, including
Ca2+ -activated, weak and strong inward-rectifying, delayed
rectifiers, voltage-gated, and L-type transient K+ channels
(286). The relative conductance of K+ channels for ammonium ions is approximately 10-20% compared to the conductance of K+ (286). Using the competitive K+ channel
blocker barium (Ba2+ ), an inhibition of the ammonia excretion in the mammalian proximal tubule was observed (1695).
Also in invertebrates K+ channels seem to be involved in
ammonia transport. Studies employing isolated perfused gills
of the green shore crab C. maenas showed that inhibition
of basolateral K+ -channels by Cs+ partially blocked both
gradient driven and active ammonia excretion (1946). In contrast, in the midgut of the tobacco hornworm M. sexta basolateral application of Ba2+ showed no effect on the active
ammonia uptake, although a ∼40% inhibition of the parallelmeasured potassium-dependent short-circuit current (ISC )
indicated Ba2+ -sensitive K+ channels to be present
(1943).
Aquaporins The protein family of aquaporins facilitate
first and foremost the transmembrane transport of water
(949). However, for some members of this family, permeability for NH3 also has been demonstrated. Studies on AQP1
employing a Xenopus oocyte expression system suggested
that AQP1 mediates an increased membrane permeability for
NH3 (1294). AQP1 function as a NH3 transporter, however,
was not confirmed by other investigators employing Xenopus oocytes and yeast complementation assays (788, 836). In
contrast, ammonia-mediating capabilities were confirmed for
four other members of the aquaporin family, namely AQP3,
AQP7, AQP8 and AQP9 (1076). All of these transporters
increase, when expressed in Xenopus oocytes, the membrane
permeability for NH3 but also for the traceable ammonia analog methylammonia (MA) (788, 1603). In AQP8-knockout
mice, minor changes of hepatic ammonia accumulation, renal
excretion of infused ammonia and intrarenal ammonia concentrations were reported (2052). For a recent review on
ammonia-transporting aquaporins and their tissue distribution in mammals, see Litman et al. (1076).
Rh-glycoproteins and Amts In 1994, Marini and others discovered that methylammonium/ammonium permeases
(MEPs) are functioning as ammonium transporters (1156).
In 2000, the same group demonstrated that the Rh (Rhesus)
proteins that were expressed in red blood cells and other
tissues in humans also transport ammonia and are analogs
of MEP and Amt proteins (1155). A thorough analyses of
the Amt/MEP/Rh family of proteins has been conducted, for
example, by Huang and Peng and Kitano and others (809,
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944). In bacteria and plants, “ammonium transporters” are
abbreviated Amts, whereas in yeast they are termed MEPs
(methylammonium permeases). As pointed out by Huang
and Peng, Rh proteins are phylogenetically related to Amt’s
with ∼14% identity between the amino acid sequences (809).
The ammonia-transporting proteins RhAG, RhBG and RhCG
(for non-human species Rhag, Rhbg and Rhcg) are glycosylated and part of a group of Rh-50 proteins (“50” for
the approximate molecular weight in kDa). Although there
are more members belonging to the Rhesus protein family
(e.g., Rh30), ammonia transport capabilities have been shown
only for the glycosylated forms so far. The debate regarding transport specificity of members of the Amt/MEP/Rh
family, however, is still ongoing. Whereas structure analysis
and biochemical assays of purified and reconstituted AmtB
transporter strongly suggest that the gaseous form NH3 is
transported (915, 916), functional expression studies of vertebrate Rh-proteins differ and are not clear-cut as to the exact
molecular species NH3 or NH4 + that is transported. When
expressed in HeLa cells, human erythroid RhAG appears to
transport both species (88). It was further shown that RhAG
transport the ammonia analog methylammonia (MA), and
that this transport is pH dependent and electroneutral (1968).
RhAG expression in humans is mainly in red blood cells
and erythropoeitic tissues, whereas the non-erythroid Rh proteins are expressed in other tissues (e.g., RhBG in kidney,
liver, GI tract, ovary and skin, and RhCG in kidney, central
nervous system, GI-tract, skeletal muscles and testes [693,
1083, 1084, 1956]). In mammals, RhBG/Rhbg is localized
to the basolateral membrane of epithelia cells (1494, 1909).
For this transporter as well, it is not clear which species of
ammonia, NH3 or NH4 + , is transported. Some studies suggest
that RhBG/bg mediates an electroneutral NH4 + /H+ transport
(1111, 1292), whereas other investigations point to a transport
of the uncharged form NH3 (1111, 1145, 2075). RhCG/Rhcg
is mostly localized apically, but not as strictly as RhBG/Rhbg
is localized to the basolateral membrane. Whereas Rhcg is
found exclusively in apical membranes in the mouse kidney
(487, 1909), RhCG shows both apical and basolateral expression in the human kidney (691). Also the transport specificity
for RhCG/Rhcg is ambiguous and ranges from a transport
that mediates both NH3 and NH4 + (62) to an electroneutral
transport of NH3 , likely by H+ / NH4 + exchange (1111). Most
recently the X-ray crystallographic structure of human RhCG
was revealed, showing that RhCG forms a trimeric complex
and promotes the passage of NH3 . Transport of NH4 + was not
precluded, although the crystallographic structure of RHCG
predicts the exclusion of NH4 + transport due to the protein’s
hydrophobic lumen (672). Mammalian Rh-proteins have been
reviewed extensively (1958) but molecular evidence for several other Rh-protein related proteins has also been found
in non-mammalian species. A phylogenetic genetic analysis
revealed that Rhag, Rhbg and Rhcg are exclusively expressed
in vertebrate species, whereas RhP1/RhP2 are predicted to
be phylogenetically older and are found in invertebrates
(809).
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Osmoregulation and Excretion
In teleost fish so far, five Rh-glycoproteins have been
identified: Rhag and two subforms of Rhbg (Rhbg1, Rhbg2)
and Rhcg (Rhcg1, Rhcg2) (1289, 1297). Expression for Rhag
was found in red blood cells (RBC), spleen and kidney and
in the membranes of branchial pillar cells (1289). Rhbg was
found to be expressed in multiple tissues including RBC,
brain, eye, heart muscle, intestine, kidney, liver skeletal muscle, skin, spleen and the gill epithelium (814, 1297). A study
in pufferfish (Takifugu rubripes) gills revealed that Rhbg in
fish is also localized to the basolateral membrane (1289). At
least in T. rubripes Rhcg1 is localized to the apical membrane of MR cells at the base of the gill lamellae, whereas
Rhcg2 is localized to the apical membrane of branchial pavement cells (1289). A functional expression study in Xenopus oocytes employing Rh-proteins cloned from trout gill and
using the ammonia analog MA showed that all five fish ammonia transporters mediate ammonia transport. That ammonia is
indeed transported by pufferfish Rhag and Rhcg2 was confirmed by using NH4 + -selective microelectrodes (1300). The
importance of Rh-proteins was also shown in the developing
zebrafish Danio rerio. Morpholino knockdown experiments
revealed a ∼50% reduction of total ammonia excretion in
the fish upon silencing of each individual transporter including Rhag, Rhbg and Rhcg2 (191). In elasmobranchs, expression of only one Rh-isoform (Rhbg) was confirmed. In the
little skate Leucoraja erinacea, Rhbg mRNA was found in
low volumes in intestinal sections, but at high expression
levels in the rectal gland and kidney (34). Most recently
Nakada et al. discovered methylammonia transport capability
for Rhp2, another glycolsylated protein belonging to the Rhlike ammonia transporter family found in the banded hound
shark, Triakis scyllium. This protein appears to be expressed
in the second and fourth loop of the sinus zone of the elasmobranch’s kidney, where it is localized to the basolateral, interstitium facing membrane. The function of Rhp2 is unclear,
although it may be involved in renal ammonia reabsorption
(1290).
Besides the studies of fish noted above, mRNA expression of Rh proteins was also confirmed in the ammoniatransporting gills and pleopods of a variety of different haline
crustaceans, among them the stenohaline marine crab Cancer irroratus (GenBank accession: AY094179), the euryhaline crabs C. sapidus (GenBank accession: AY094178) and
C. maenas (1950), the isopod Idotea baltica (GenBank accession: AY094181) and the true freshwater crab Dilocarcinus
pagei (GenBank accession: AY094180). So far in crustaceans
only one Rh isoform has been identified. In the tobacco hornworm Manduca sexta, mRNA expression analysis revealed
a moderate abundance of an Rh-like protein in the midgut,
trachea and fatbody and high expression levels in the hindgut,
ganglia and the Malpighian tubules (1943). The cellular localization of arthropod Rh-like ammonia transporters has not
been determined yet (Fig. 34B-8). In addition to findings in
the tobacco hornworm, Rh-proteins have been identified in
other insects as well. In the yellow fever mosquito Aedes
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Comprehensive Physiology
aegypti, two Rh-isoforms have been identified, Rh50-1 and
Rh50-2. Both are expressed in the ammonia-excreting anal
papillae of the freshwater-inhabiting larvae and respond to
increased environmental ammonia levels (1948).
Interestingly, in insects and other invertebrates, for example C. elegans, a genome analysis revealed the presence of
Amt transporters also. Although the function of the Amts in
animal systems as ammonia-transporting proteins have not
been investigated yet, these Amts group together with functional Amts identified in plants (1948) and critical amino
acids in the pore center (H168 and H318 in E. coli AmtB) are
conserved, at least in the insect Amts.
The gene expression data base for Drosophila
melanogaster (FlyAtlas [285]) indicated that the Rh-protein
and Amt of the fly are expressed in the same tissues, however, in most cases with complementary mRNA expression
levels. While Rh50 in the fruit fly D. melanogaster was
highly expressed in neuronal tissues, Malpighian tubules and
hindgut, mRNA levels of Amt were low in those tissues.
In contrast, in the salivary glands, Amts showed increased
expression levels, while low expression levels were detected
for Rh50.
In-situ hybridization studies in the freshwater planarian
S. mediterranea indicated that an identified Rh-like ammonia
transporter is strongly expressed in the epidermal epithelium.
Together with responses of mRNA expression levels of this
transporter upon changes of environmental ammonia or pH
(1947), this suggests a role of the epidermal Rh-protein in
ammonia excretion processes.
V-type H+ -ATPase and ammonia trapping Although
not capable of transporting ammonia by itself, the V-type
H+ -ATPase plays a crucial role in transepithelial ammonia
excretion and uptake processes. Membrane diffusion of NH3
strictly depends on the transmembrane NH3 partial pressure
gradient (PNH3 ), often generated by the action of the VATPase or cation/H+ exchanger. The action of both transporters is responsible for lowering locally the pH on one
side of the membrane and generating thereby a transmembrane PNH3 , a mechanism referred as “ammonia trapping”
(Fig. 34B). The transport of ammonia via Rh glycoproteins can also be energized by the proton pump, regardless of whether the Rh-proteins function as NH3 channels
or H+ /NH4 + exchangers. In freshwater fish, the importance
of the V-ATPase in ammonia excretion processes has been
described extensively (e.g., 191, 1665, 1953, 2007, 2041).
For example, eliminating the low pH in the apical unstirred
gill boundary layer by setting the pH to 8 using HEPES caused
a significant reduction in ammonia efflux rates in the rainbow
trout Oncorhynchus mykiss (2007). In zebrafish (Danio reio),
larval ammonia extrusion via the skin depends directly on
the presence and action of a H+ -ATPase, as demonstrated by
reduced ammonia excretion rates in H+ -ATPase knockdown
animals and the inhibitory effect of bafilomycin A1, a specific
inhibitor for the V-Type H+ -ATPase (163, 1665).
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Comprehensive Physiology
Also in invertebrates, a V-type H+ -ATPase linked to
ammonia transport has been demonstrated. In the green
shore crab C. maenas, branchial active ammonia excretion
was reduced by 66% after blocking the proton pump with
bafilomycin A1. It is noteworthy that, in contrast to freshwater fish, the H+ -ATPase in the crab gill epithelium is localized
to vesicles rather than the apical membrane (1954). Further,
the active ammonia uptake in the midgut of the tobacco hornworm M. sexta was significantly reduced after bafilomycin
A1 application, indicating participation of a V-ATPase in the
intestinal ammonia uptake mechanism (1943).
In the kidney of rats, expression of the apical ammonia transporter Rhcg seems to be colocalized with the
presence of an apical H+ -ATPase. In cortical collecting
ducts, H+ -ATPase-rich intercalated cells showed parallel apical Rhcg abundance, whereas H+ -ATPase-negative principal cells showed only a faint Rhcg expression. In the outer
medulla and the upper portion of the inner medulla, Rhcg
abundance was limited to a subpopulation of cells within the
collecting duct with an apical H+ -ATPase, indicating that
Rhcg expression in medullary collecting ducts is linked to the
action of an apical proton pump (487).
Vesicular ammonia transport In the gills of the green
shore crab C. maenas, gradient-driven or active ammonia
excretion was substantially inhibited (∼60-100%, depending on the experiment and drug) after the application of
microtubule inhibitors such as colchicine, taxol and thiabendazol (1954). This finding led to the suggestion of a vesicular
ammonia-trapping mechanism in which cellular NH3 moves
via membrane diffusion or through Rh-proteins into acidified
vesicles to be transformed into its membrane-impermeable
ionic form, NH4 + . For directed excretion these NH4 + -loaded
vesicles would then be transported to the apical membrane
for exocytotic release. A similar intracellular ammonia transport, which depends on a vesicular H+ ATPase and an intact
microtubule network, was also described for the active ammonia uptake in the midgut epithelium of the terrestrial tobacco
hornworm Manduca sexta (1943) (Fig. 34C).
Osmoregulation and Excretion
many different tissues including the endothelium of the arterial vasa recta (525) and extra-renal tissues like bone marrow,
spleen, fetal liver, GI tract, ureter and bladder and astrocytes
(2051).
Evidence for urea transporters have also been found in
tissues of non-mammalian vertebrates. Examples include the
renal tissues of fish (1238) and amphibians (1094, 1628), the
ventral skin of toads (213, 468, 478), the gills of teleost fish
(1239, 1927, 1928) and the gills, rectal gland, kidney and
intestine of elasmobranchs (34).
In the gills of the spiny dogfish, evidence was found for a
secondary active Na+ -urea exchanger energized by the basolateral Na+ /K+ -ATPase and localized to the basolateral membrane. This phloretin-sensitive urea transporter serves most
likely to retain urea in elasmobranchs where high plasma urea
concentrations are maintained for osmotic homeostasis (531).
In addition, studies on brush border membrane vesicles from
kidney tissues of the little skate Raja erinacea revealed evidences for two additional types of UTs: a phloretin-sensitive,
non-saturable uniporter in the dorsal section and a phloretinsensitive, sodium-linked urea cotransporter in the ventral
section of the kidney (1249). Sequence information of the
expressed UT mRNAs has not been linked yet to the different kind of UTs in elasmobranchs. UT transcripts, termed
efUT-1, efUT-2 and efUT-3, have recently been identified in
the holochephalan elephant fish, Callorhinchus milii. efUT-1
is orthologous to the elasmobranch UTs, whereas efUT-2
and efUT-3 are novel UTs. When functionally expressed in
Xenopus oocytes, all UT transcripts, including the short and
long variants of efUT-1 and efUT-2, induced increases in
14
C-urea uptake of more than 10-fold. Phloretin sensitivity
of urea uptake suggested that the identified UTs are facilitative transporters (877). The physiology of urea transport in
fish has been reviewed extensively by McDonald and others
(1202). Information gained from Expressed Sequences Tags
(EST), for instance from the blue crab Callinectes sapidus
(GenBank accession #: CV527852) or the jewels wasps Nasonia vitripennis (GenBank accession #: XP_001601729), indicate that urea transporters are also expressed in invertebrate
tissues.
Urea Transporting Proteins and Mechanisms
Aquaporins
The urea transporters (UT) play a crucial role in urine concentration and urea recycling in the mammalian kidney. All UTs
promote a facilitated urea transport (445, 525, 2051). UTs
belong to two different subfamilies encoded by two different genes, UT-A and UT-B. Several UTs belong to the UT-A
family. UT-A1 is expressed exclusively in IMCD cells and its
activity is regulated by vasopressin (AVP). UT-A2 is localized to the lower portions of the thin descending limbs of short
loops of Henle, whereas UT-A3 is restricted to the terminal
IMCD, where it is localized to both the apical and basolateral
membranes. UT-A4 and UT-A5 were detected extrarenally in
heart and testis, respectively. UT-B has been found in RBC and
Some mammalian aquaporins, mostly in the subclass of
aquaglycoproteins (GLPs), transport organic compounds such
as glycerol and urea in addition to water (698, 964). Ureatransporting GLPs have been identified among them, specifically AQP3, AQP7, AQP9 and AQP10. For a recent review
on urea-transporting aquaporins, their tissue distribution and
co-localization with urea transporters in mammals, see also
Litman and others (1076, 2003). For non-mammalian species,
urea permeability has not been shown to date, most likely
because it has not been investigated yet. Molecular analysis and functional expression studies of three cloned AQPlike proteins (HC-1, HC-2 and HC-3) in the gray tree frog
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Osmoregulation and Excretion
Hyla chrysoscelis revealed that at least one of them, HC-3,
exhibits glycerol transport capabilities and shows a very high
degree of nucleotide identity (> 80%) to the human ureatransporting aquaglycoprotein AQP3 (2076). One can predict
that the amphibian aquaporin-like protein HC-3 also mediates
urea permeability.
Uric Acid Transporting Proteins and Mechanisms
With a pKa1 = 5.75, uric acid behaves as a weak acid and
occurs predominantly (∼98%) as urate anion at physiological
pH. The urate anion cannot readily pass through cell membranes, and for epithelia, excretion transporters are required.
By far the most literature on urate transporters can be found on
mammals, through to the role of urate transport in diseases like
gout (1724). In mammals, nearly all plasma urate is filtered
by the renal glomeruli; however, close to 90% of the initial filtrate is reabsorbed in the proximal tubule back into the blood
stream. Beside renal excretion, urate is also excreted by hepatocytes into the bile canniculi and into the gastrointestinal
tract. A key player for urate reabsorption is the apical localized
URAT1, which is proposed to transport intracellular organic
anions such as lactate in exchange for urate (496, 1609, 2033).
It is speculated that URAT1 is functionally coupled to apical
sodium monocarboxylate cotransporters, SMCTI and SMCT2
(827). Mediated by the Na+ /K+ -ATPase-powered, inwardly
directed Na+ gradient, both proteins transport lactate into the
cells, which can in turn be utilized by URAT1. Another transporter involved in renal urate reabsorption is the apical localized organic anion transporter-4 (OAT4), which acts as a lowaffinity urate (anion)/dicarboxylate exchanger (47, 684). For
basolateral exit, three transporters with urate transport capabilities have been proposed: (i) the organic anion transporter1 (OAT1) (828, 1647); (ii) the organic anion transporter-3
(OAT3), which functions as a urate-decarboxylat exchanger
(61); and (iii) possibly the most important transporter, the
glucose transporter-9 (GLUT9), which transports urate 45- to
60-fold faster than glucose/fructose (264) and functions as a
high-capacity/low-affinity urate transporter (1915). For apical urate excretion, several transporters have been discussed,
including a splice variant of GLUT9 (GLUT9N), the sodium
phosphate transporters-1 and -4 (NPT1, NPT4), the voltagedriven organic anion transporter, OATv1 and the ATP-binding
cassette transporter, MRP4. GLUT9N shows apical expression (54); however, urate transport capacities have not been
shown yet. In contrast, for OATv1, NPT1, NPT4 and MRP4
urate transport capability has been demonstrated (48, 1880,
1896). Aside from its apical expression in renal cells, MRP4,
which mediates an ATP-dependent urate transport, it is also
found basolaterally in liver cells and is therefore suggested to
be responsible for hepatic urate transport into the circulatory
system (1896). Very little is known about urate transporters in
non-mammalian systems. However, genome and EST projects
on a wide variety of other systems including amphibians, fish,
insects, crustaceans and nematodes will certainly promote
future studies regarding this subject.
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Nitrogen Excretion of Taxonomic Groups
With some exceptions, aquatic invertebrates, teleost fish and
amphibian larvae are ammoniotelic, excreting the majority
of their nitrogenous waste products in the form of ammonia.
Adult amphibians, mammals and marine elasmobranchs are
in general ureotelic, with the main excretory waste product
being urea. Most adult insects, arachnids, birds and reptiles are
uricotelic, excreting predominately uric acid or other purinebased compounds like guanine, allantoin and xanthine. Often,
however, depending on the lifestyle or the particular stage of
life history, a single species might switch from one main
excretory product to another as seen, for example, in amphibians. For animals, our knowledge of excretory mechanisms of
any given nitrogenous waste product is still very limited. So
far, information on excretory processes and mechanisms has
been gained usually by performing flux studies and studying the effects of transporter-specific inhibitors. However,
over the last two decades or so, powerful molecular techniques such as functional gene expression analysis of putative
transport molecules and quantitative mRNA, as well as protein expression analysis and immunohistochemistry, also have
been employed. The vast majority of these studies, including
molecular approaches, have been performed in mammals and
fish and were covered extensively in recent years (191, 813,
1112, 1205, 1289, 1468, 1665, 1953, 1959, 2032, 2041). An
extensive review on nitrogen excretion in invertebrates has
been provided by O’Donnell (1338) and thus will only be
updated here.
Protozoa, porifera and cnidaria Studies on nitrogen excretion in free living protozoa, porifera and cnidaria
are sparse. The free living freshwater microflagellates,
Monas sp., exhibit average ammonia excretion rates of 0.76 to
1.23 μmol of NH4 + per mg (dry wt) per hr (1664). So far, the
mode of ammonia excretion in protozoa is unclear. Freshwater protists, such as Amoeba proteus, osmoregulate by means
of a contractile vacuole. In the membranes of this contractile
vacuole high abundance of a V-type H+ -ATPase was detected
(1315). The proton-pumping action of this ATPase will likely
generate a cytosol to vacuolar H+ gradient and thereby also
a PNH3 , promoting ammonia trapping via membrane diffusion. This NH3 movement might be supported by a so far
unidentified Rh-protein within the same membrane. Therefore, it is conceivable that ammonia waste in freshwater protists is excreted along with excess water by the action of the
contractile vacuole. Contractile vacuoles have also been found
in freshwater sponges, here in amoebocytes, pinacocytes and
choanocytes (181). For the symbiotic sponge Haliclona cymiformis, an ammonia excretion rate of 4.6 μg ammonium-N g−1
dry mass h−1 was described. Interestingly, ammonia released
by the sponge serves as an important nitrogen source for
its symbiotic partner, the rhodophyte Ceratodictyon spongiosum (398). This kind of symbiotic partnership was also
suggested for the giant sea anemone Entacmaea quadricolor
and its symbiotic zooxanthella partner. In fact, at times of
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Comprehensive Physiology
Osmoregulation and Excretion
high demand of nitrogenous compounds by the algae during
daytime, E. quadricolor exhibits an ammonia uptake, likely
waste ammonia from the anemoefish Amphiprion bicinctus.
At nighttime, when metabolic demands of the algae are minimal, the rate of ammonia uptake by the sea anemone decreases
to near zero (1575). In contrast, aposymbiotic sea anemones
(lacking zooxanthella) Aiptasia pulchella excrete ammonia
during daytime (1995). Mechanisms of ammonia uptake or
excretion in cnidaria are largely unknown. A genome project
revealed that the starlet sea anemone Nematostella vectensis does likely express a Rhesus-like ammonia transporter,
exhibiting ∼44% identity in its amino acid sequence to the
ammonia-transporting mammalian Rh-glycoproteins. In addition, genes for several putative AMT-like ammonia transporters have been identified in this species.
lower expression also in other cell types (851). These findings suggest that ammonia is excreted, at least partially, via the
integument.
Knock-down experiments revealed that CeRh2 is not
essential for embryonic development, while silencing CeRh1
caused a lethal phenotype mainly affecting late stages of
C. elegans embryonic development (851). In addition to the
Rh genes, C. elegans also expresses 4 AMT-like ammonia
transporters (amt-1 to amt-4; see http://www.wormbase.org.).
While no information is available to date for expression patterns of amt-1, amt-2 and amt-4, amt-3 is expressed in adult
nematodes in the head nerves, ring and tail nerves and in the
intestine. Expression in the nerve tissues might serve protective purposes, while abundance of amt-3 in the intestine
might facilitate ammonia excretion or ammonia uptake. However, verification that amt-proteins expressed in animal tissues
indeed promote ammonia transport must still be obtained.
So far little is known about nitrogen excretion in planarians. A most recent study on the freshwater planarian
Schmidtea mediterranea revealed that ammonia is excreted
across the mucous epidermis in an active fashion at a rate
of 0.70±0.03 μmol−1 gFW−1 h−1 (1947). This excretion
appeared to be dependent on the environmental pH and
inhibitor experiments combined with gene-expression studies indicated further a number of transporters involved in the
ammonia excretion mechanisms that include the V-ATPase,
Na+ /K+ -ATPase, cation/H+ exchangers, the carbonic anhydrase and a Rh-like ammonia transporter. According to a
proposed working model (Fig. 35), interstitial fluid ammonia is pumped as NH4 + across the basolateral membrane by
Nematoda, planaria and annelida
Parasitic nematodes such as Ascaris lumbricoides (1960),
Trichinella spiralis larvae (713) and Nematodirus sp. (1569)
excrete ammonia as their primary waste product. Similarly, the free-living nematode Caenorhabditis briggsae also
excretes predominately ammonia but no significant amounts
of urea or uric acid (1580). Possible excretion routes include
the integument, the nephridial system or the gut. In the genetic
model system, Caenorhabditis elegans genes for two Rh
homologues, CeRh1 and CeRh2, have been identified that are
highly conserved and similar to the human Rh-glycoproteins
(851). A transgenic analysis using reporter genes showed
that CeRh1 is mainly expressed in hypodermal tissue, with
Fresh water
Epithelium
Plasma
Boundary
water
Bulk water
pH = 5.5-6.5
CO2
CO2
CO2
CA
HCO3–
+
H+
HCO3– + H+
Rh
Rh
NH3
NH3
NH3
+
H -ATPase
NH4+
NH4+
ATPase
?
NH4+
Na+
–
NH3 + H+
Na+
NHE
NH4+
H+
H+
Na+
NH3
Basolateral
Apical
Figure 35 Ammonia excretion across the epidermal epithelium in the freshwater planarian
Schmidtea mediterranea. The proposed mechanism is described in the text. The figure is
modified after (1947).
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Osmoregulation and Excretion
Na+ /K+ -ATPase into the cytoplasm. Protons provided by a
cytoplasmatic carbonic anhydrase are then excreted apically
via the V-ATPase and a cation/proton exchanger (NHE), acidifying the unstirred boundary layer to a pH of approximately
5.5-6.5.
The generated low pH protonizes apical NH3 into its ionic
form NH4 + and thereby creates a transcellular PNH3 gradient.
It is proposed that an Rh-like ammonia transporter mediates
the NH3 transport across the apical cell membrane. Due to fact
that in freshwater organisms epithelia facing the environment
generally show a very low ion conductance to avoid passive
effluxes along a steep osmotic gradient (299, 1942, 1945),
it is therefore assumed that paracellular NH4 + diffusion is
negligible. Interestingly, the ammonia excretion mechanism
in this flatworm reveals striking similarities to the current
model suggested to be in function in gills of freshwater fish
(see below).
Animals belonging to the phylum of Annelida are usually ammoniotelic. Ammonia excretion rates of the marine
and osmoconforming lugworm Arenicola marina were measured as ∼33 μmol g dry weight−1 day−1 (calculated from
[1519]). This excretion was salinity dependent, and an acute
reduction of the environmental salinity to 16 resulted in
a strong increase of the excretion rate. After its maximum
between 12 hrs and 36 hrs after exposure the excretion rate
gradually decreased to levels only slightly above the control rates. This pattern was mirrored in the parallel oxygen
consumption rates. Varying ammonia excretion rates were
related to amino acid-based extra and intracellular volume
regulation in this annelid (1519). Also the freshwater living
medicinal leech Hirudo medicinalis excretes predominantly
ammonia (1870) after ingestion of serum. Excretion rates
rapidly increased to a peak of ∼30 μmol ammonia g FW−1
day−1 within a day of feeding, with a second peak starting at day 5-7. After day 15, post-feeding excretion rates
dropped slowly to zero over the next week. Interestingly,
serum feed H. medicinalis also exhibits a peak of urea excretion one day after feeding (∼14 μmol urea/ animal/day),
which dropped to values less than 2 within the following
day. The early ammonia and urea excretion peaks probably
result from the elimination of ammonia and urea ingested
with the serum, which contained 2.2 mM ammonia and 6.6
mM urea. Ammonia excretion rates of the second phase are
very likely due to the digestion of serum proteins. Symbiotic microorganisms do not significantly contribute to ammonia formation (1870). Not much is known about ammonia
excretion mechanisms in Annelida. The polychetous annelids
Nereis succinea and Nereis virens actively excrete ammonia.
Active ammonia excretion by the polychetous annelids N.
succinea and N. virens depends partly on the activity of the
Na+ /K+ -ATPase and can be inhibited with ouabain by 30%
and 60%, respectively (1149). The medicinal leech H. medicinalis osmoregulates via the integument, exhibiting an active
amiloride sensitive Na+ uptake that is driven by the Na+ /K+ ATPase (1940, 1941). The skin is further coated with a mucus
layer, providing an apical unstirred boundary layer (306). It is
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Comprehensive Physiology
conceivable that ammonia excretion occurs via the integument by utilizing transporters involved in osmoregulatory ion
uptake. Additional excretory pathways via the metanephridial
system or the intestinal tract, e.g. the Na+ /K+ -ATPase energized caecum (1222), might play also a role. Other possible
tissues for ammonia elimination in annelids are the wellvascularized parapodia of marine polychaetes as pointed out
by O’Donnell (1338). Due to their primary function in locomotion, the tissue is well ventilated and therefore also used
for respiratory gas exchange. Consequently, the transepithelial hemolymph to environment ammonia gradient is very
likely favorable for ammonia excretion in animals inhabiting
the free water column (1338). The terrestrial living earthworm
Lumbricus terrestris, an oligochaete, excretes predominantly
ammonia while feeding and water supply is not limited. It
becomes ureotelic, however, when fasted or when water supply is limited (1117). Ammonia excretion occurs here likely
via the gut, where extremely high ammonia concentrations
(11-104 mM) can be detected (1830). Similar to H. medicinalis, an amiloride-sensitive Na+ transport across the skin was
documented in L. terrestris as well (979). Therefore, the possibility of NH4 + excretion via the skin cannot be excluded. In
contrast to the pathways of ammonia excretion, urea is eliminated via the metanephridial system (1830). It is noteworthy
that in L. terrestris all enzymes for a functional urea cycle
have been identified (140).
Ecinodermata
Echinoderms are ammoniotelic. After collection from the
sea, and assumed to have recently fed, the sea urchin Tripneustes gratilla excretes ammonia at rates of ca. 0.5 μmol g
dry weight h-1. This rate is similar to rates found in collected horned sea stars Protoreaster nodosus and the brittle
star Ophiorachna incrassate. Ammonia excretion in these
three different echinoderms seems to depend on the time
of the day. While oxygen consumption rates remained similar, ammonia excretion rates decreased in animals collected during nighttime (466). Investigations by Sabourin
and Stickle on the brackish water tolerant sea cucumber Eupentacta quinquesemita and sea urchin Strongylocentrotus droebachiensis showed ammonotely for both
species in full-strength seawater, with minor excretion of
urea (∼2-3% of total N-excretion) and primary amines
(2-4% of total N-excretion). Apparently nitrogen excretion
rates are salinity dependent in sea cucumber but not in sea
urchin.
While in Eupentacta quinquesemita, ammonia excretion
rates are reduced by ∼50% when acclimated to 20 and 15
salinity, excretion rates of primary amines increased approximately 4-fold and 10-fold, respectively. In both species, urea
excretion rates remained low and did not changed in response
to changing salinities (1592). One can speculate that in low
salinity, primary amine synthesis is enhanced for the purpose of intracellular volume regulation and that increased
excretion/loss of these energetically valuable molecules is
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Comprehensive Physiology
rather a negative side effect of the salinity stress handling
than for the purpose of nitrogen excretion per se. A genome
project, supported also by expressed sequence tags, on the
California purple sea urchin, Strongylocentrotus purpuratus
indicates that echinoderms also express Rh-ammonia transporters (GenBank accession #: XP_789738, XM_784645)
and an urea transporter-like protein (GenBank accession #:
XM_781359). To date it is not known in which tissues the
Rh-protein and urea transporter are expressed, or their specific role in ammonia and urea excretion.
Mollusca
Cephalopods are quite active compared to others molluscs.
These carnivorous animals excrete the majority of their
nitrogenous waste as ammonia, with the ctenidia playing
a significant role in excretion (1473). Pelagic squid, such
as Illex illecebrosus are in general more active than bentic
octopuses and excretion rates in these animals are high (1.4
mM NH4 + /kg/hr) at rest but increase about fivefold during
exercise (776). Similar excretion rates were also reported for
Loligo forbesi, an active nektonic predator (158). In contrast,
ammonia excretion rates in Octopus rubescens were about
five times lower (at rest) when compared to plelagic squid
I. illecebrosus (776).
In addition to ammonia, both squid and octopus also
excrete considerable amounts of their nitrogenous waste in
form of urea. For instance, at rest, urea accounts for 24% and
30% of total nitrogen excretion in the squid Illex illecebrosus and Octopus rubescens, respectively (776). The mode of
ctenidial ammonia excretion is so far unknown, but ammonia
excretion via the kidney is thought to occur by acid trapping,
as reported for Octopus dolfeini (1473). The pericardial fluid
in this octopus is slightly alkanline and contains lower total
ammonia concentrations than does the blood (1473). In addition to excretion, in squid, ammonia is also used for buoyancy.
These ammoniacal animals sequester ionic NH4 + , which is
lighter than, for example, Na+ , Ca2+ , Mg2+ or SO4 2− , either
in specialized “coelomic chambers,” observed in squid of the
family Cranchiidae, or in the muscle tissue (other ammoniacal squid). Here the mantle and arms are interspersed with
vacuoles that most likely contain ammonium. Ammonium
concentrations in these specialized organelles are very high,
ranging between 300 and 500 mmol l−1 (302, 2040). Considering the toxicity of ammonia, it is noteworthy that in ammoniacal squids the body mass is comprised of approximately
50-60% ammonium fluid (302).
Lamellibranchs are also ammoniotelic. Interestingly, they
also excrete considerable amounts of their nitrogenous ballast in the form of amino acids. For instance, sand-mussels
Donax serra and D. sorididus excrete 70% and 73% of their
total dissolved nitrogen as ammonia and 30% and 22% in
the form of amino acids, respectively (325). Since bivalves
do not possess biochemical pathways to detoxify ammonia
into less toxic urea or uric acid (141), the excretion of these
nitrogenous compounds is negligible, with excretion rates of
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Osmoregulation and Excretion
less then 1% of total nitrogen release (325). In contrast to
marine species, freshwater bivales are sometimes challenged
by extended periods of air exposure during droughts of unpredictable lengths. A study on the freshwater clam Corbicula
fluminea showed that during air exposure, hemolymph ammonia levels remain low at pre-emersion levels (60 to 140 μM)
for about 4 days before they dramatically increase over the
next 3 days to 8 mM, resulting in death of the animal. The data
suggest that Corbicula fluminea as a representative of freshwater bivalves appear to be able to suppress protein catabolism
during the first few days of emergence to avoid toxic ammonia
accumulation (237). Not much is known about the ammonia
excretion mechanism in bivalves. A study by Mangum and
others in euryhaline Ragia cuneata suggests that ammonia
is actively excreted. Inhibitory effects on ammonia excretion rates employing oubain suggest the participation of the
Na+ /K+ -ATPase in this process, as shown for other aquatic
invertebrates as well (1149, 1946). Similar to the lamellibranchs, marine prosobranch snails also do not excrete urea
(457). In Thais (Nucella) lapillus, ammonia excretion was
found to be salinity dependent, with significantly lower rates
detected in low salinity (17.5 ppt salinity) when compared
to higher salinities (1763). Regardless of the environmental
salinity and temperature, these snails excrete/lose also primary amines. However, when expressed as a percentage of
ammonia excretion rates, in low-salinity environments the
loss of primary amines is higher than the loss in higher salinities (1763). Intertidal polyplacophora such as Chiton pelliserpentis are also ammonotelic; they do, however, excrete
high amounts of urea. In high shore Chitons, which presumably are exposed to air for prolonged time periods, urea-N
can make up to 34% of combined ammonia and urea nitrogen, whereas urea-N excretion rates in low shore Chitons are
lower (798). In contrast to their acquatic relatives, terrestrial
snails are uricotelic/purinotelic, excreting their nitrogenous
waste predominantly as uric acid, guanine and xanthine (458,
850, 1854). For instance, in the kidney contents (excreta) of
the giant South American snail Strophocheilus oblongugus,
70% of total N accounted for purinic compounds, with uric
acid being the most prominent (∼64%), followed by xantine (∼24%) and guanine (12%) (1854). In the garden snail
Helix pomatia, almost all (90%) of the nitrogen is excreted in
the form of purines (850). In addition to purinic compounds,
shelled snails also excrete gaseous NH3 through the shell at
rates between 1.0 μmol and 0.01 μmol NH3 /g live wt/day.
Shell-less slugs, in contrast, absorb NH3 from the atmosphere
(1090).
Crustacea
The main site for ammonia excretion by aquatic crabs is the
phyllobranchiate gill (307, 963, 1516), featuring a single-cell
layered epithelium covered by an ion-selective cuticle (56,
1049, 1380, 1954). Several transporters and enzymes putatively linked and involved in ammonia transport have been
shown to be present in the branchial epithelium of crabs,
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Osmoregulation and Excretion
among them the Na+ /K+ -ATPase, cation/H+ -exchanger,
K+ -channels, Na+ -K+ -2Cl− cotransporter, H+ -ATPase and
the Rh-proteins (1557, 1848, 1950).
Ammonia excretion in aquatic crabs Studies on isolated perfused gills of several aquatic crabs showed that
ammonia can be excreted actively against a four- to eightfold
inwardly directed ammonia gradient across both anterior and
posterior gills to a similar degree, despite their different morphological and physiological characteristics (339, 629, 1848,
1945). Under physiologically relevant conditions, the potential for active branchial ammonia excretion is significantly
greater in the marine Cancer pagurus than in freshwateracclimated Chinese mitten crabs Eriocheir sinensis. This is
remarkable since Cancer pagurus gills have a much larger
ionic conductance (∼250-280 mS. cm−2 ) compared to that
of Eriocheir sinensis gills (∼4 mS. cm−2 ). It is noteworthy that posterior gills of Carcinus maenas, thought to play
the dominant role in osmoregulatory NaCl uptake, and also
anterior gills, thought to be primarily responsible for gas
exchange, are equally capable of active ammonia excretion
(1945).
In the blue crab Callinectes sapidus, ammonia excretion
rates are correlated with Na+ absorption (1482). The same
result was obtained for both the Chinese crab Eriocheir sinensis (1419) and the shore crab Carcinus maenas (1106). In the
marine crab Cancer pagurus, active branchial excretion of
ammonia is completely inhibited by ouabain (1945), suggesting the Na+ /K+ -ATPase is the only driving force for excretion.
However, in the gills of Carcinus maenas acclimated to brackish water, both gradient-driven (1106) and active ammonia
excretion (1946) are only partially inhibited by ouabain, indicating a second active mechanism responsible for branchial
ammonia extrusion in this species.
The presence of an apically located amiloride-sensitive
Na+ /NH4 + exchanger, transporting NH4 + from the epithelial
cell into the ambient medium in exchange for Na+ , has been
suggested for Callinectes sapidus (1482) and for Carcinus
maenas (1106, 1688). Indeed, branchial mRNA expression
of a Na+ /H+ -antiporter, putatively transporting NH4 + ions as
well, was demonstrated in Carcinus maenas (1847) and in
Eriocheir sinensis (1952).
Further studies on the branchial ammonia excretion mechanism in Carcinus maenas employing the K+ -channel blocker
Cs+ revealed that basolateral but not apical K+ -channels play
a role in the excretory process (1946). In addition, experiments
inhibiting the branchial V-Type H+ -ATPase by bafilomycin
A1 resulted in a reduction of active ammonia transport by
66%, identifying the H+ -ATPase as an additional active component in the excretory mechanism of the shore crab (1954).
While in Eriocheir sinensis a V-Type H+ -ATPase has been
localized to the apical membrane of the gill epithelium (1379),
in Carcinus maenas this pump was found predominantly in
the cytoplasm, likely associated with vesicles (1955). This
latter finding led to the suggestion (1954) of a vesicular
ammonia-trapping mechanism in which cellular NH3 diffuses
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Comprehensive Physiology
into acidified vesicles to be transformed into its membraneimpermeable ionic form NH4 + . For a directed excretion, these
NH4 + -loaded vesicles would then be transported to the apical
membrane for exocytotic release. Such an excretion mechanism was supported by data showing total inhibition of active
ammonia excretion by blockers of the microtubule network,
including colchicine, thiabendazole and taxol (1954), and the
fact that buffering of the experimental solutions by 2.5 mM
TRIS (pH 7.8) had no effect on the active ammonia excretion rates. This indicates that acidification of the subcuticular
space or the gill boundary layer has no influence on driving ammonia across the apical membrane of the gill epithelia
(1945, 1946). The resulting hypothetical model of the ammonia excretion in Carcinus maenas is described in detail in
Fig. 36.
It is likely that in crabs that utilize a proton gradient across
the apical membrane of the epithelial cell to accomplish NaCl
uptake from highly diluted media, such as the partially limnic
Chinese crab Eriocheir sinensis or the true freshwater crab
Dilocarcinus pagei, NH3 diffuses across the apical membrane
along its partial pressure gradient, as shown, for instance, in
freshwater rainbow trout Oncorhynchus mykiss (2007). As
mentioned above, mRNA expression of Rh-like ammonia
transporters have been shown in gills and pleopods of many
different crustaceans. Due to the lack of specific antibodies,
the cellular localization of this putative ammonia transporter
is unclear.
One can further speculate that in marine and brackishwater crabs, the Rh-proteins are not localized in the apical membrane of the gill epithelium, since here an apically
H+ -ATPase, which generates an outwardly directed PNH3 , is
missing. Ammonia (NH3 /NH4 + ) permeable structures would
be a disadvantage, allowing ammonia influxes when the animals are exposed to high external ammonia concentrations.
Many aquatic crab species are benthic living animals, hiding
under stones or burying themselves in the sediment for long
periods, for example during low tide or in the winter season.
Under conditions where crabs are situated at sites with low
rates of ambient water exchange plus the fact that the animals
produce and excrete metabolic ammonia, concentrations of
the ambient ammonia can reach high values.
Ammonia excretion in terrestrial crabs The mechanisms by which air-breathing crustaceans excrete nitrogenous waste into the terrestrial habitat have been investigated
intensively (642, 643, 1348, 2017, 2020). Terrestrial crabs
seems to tolerate considerably greater hemolymph ammonia loads when compared to aquatic species (1950), likely
because diluting ammonia to non-toxic levels in the urine
might require an unsustainable water loss in a land crab
(2018). In all land crabs examined to date, the gills have
become adapted for reabsorption of salt from the primary
urine directed through the branchial chamber (1254, 2017,
2019), allowing diffusive NH3 loss and NH4 + extrusion in
exchange for required ions from the urine. For land-living
Volume 4, April 2014
Comprehensive Physiology
Osmoregulation and Excretion
Carcinus maenas
Gill epithelium
NH3
RhCM
Brackish/sea water
Metabolic
ammonia
NH3 + H+
NH4+
Na+
+)H+
(NH4
Na+
K+/NH4+
NH4+
ATPase
H+
NH3
ATPase
K+/NH4+
NH4+
NH4+
NH4+
Hemolymph
Apical
Cuticle
Figure 36 Branchial ammonia excretion model proposed for the green shore crab Carcinus
maenas. The proposed mechanism is described in the text. The figure is modified after (1958).
crustaceans, different types of ammonia excretion modes have
been described so far.
A “storage-excretion” mechanism was shown in diverse
air-breathing crabs, for example in Potamonautes warreni
(1255), in Austrothelphusa transversa (1073), in Discoplax
hirtipes (416) and in Cardisoma carnifex (2022). Discoplax
hirtipes excretes 99% of its waste as ammonia, but while
breathing air, the rate of nitrogen loss in the urinary flow
is only 0.2 μmol kg−1. h−1 and NH3 is volatilized at a
very slow rate (0.4 μmol. kg−1. h−1 ). On reimmersion, the
ammonia excretion rate is transiently elevated to 1,100
μmol. kg−1. h−1 compared to the normal aquatic excretion
of 300 μmol. kg−1. h−1 . Potamonautes warreni also does not
excrete while in air, but on return to water the crab excretes
ammonia across the gills at 4.9 mmol. kg−1. h−1 as compared
to the normal rate in water of 70 μmol. kg−1. h−1 . Gill irrigation
appears to be a ubiquitous activity following excursions into
the terrestrial environment (416). The requirement to reimmerse for branchial ammonia excretion may ultimately limit
the duration of air breathing in amphibious species. However, Austrothelphusa transversa can spend many months
without access to water without the need of ammonia excretion during that time period (1073). It was suggested that the
near-cessation of nitrogen excretion in A. transversa implied
reduced nitrogen catabolism and temporary nitrogen storage.
The speed and brevity of the excretion pulse in P. warreni
during immersion showed that wastes stored during terrestrial forays are rapidly excreted on return to water. Further
Volume 4, April 2014
investigations are required to determine the storage product,
but an accessible intermediate such as glutamine is implicated. Apical H+ /NH4 + exchange and V-ATPase-driven cell
alkalization have been suggested as likely mechanisms of
transbranchial ammonia transport (1073).
Purine is stored in large amounts in connective tissue cells
throughout the bodies of some land crabs (1071). In Gecarcoidea natalis, this stored purine is normally synthesized de
novo, from excess dietary nitrogen. Recent data, including
enzyme activities and nitrogen utilization (1072), have led
to the suggestion of a storage-excretion function for the urate
accumulated by G. natalis (644). Most land crabs recycle their
urine over the branchial surfaces, producing a dilute fluid “P”
(1253, 2019). The air-breathing ghost crab Ocypode quadrata
also recycles the urine and produces “P”, which can be as
little as 10% of the osmotic strength of the primary urine. The
ammonia concentration of the primary urine of the ghost crab
is extraordinarily high. For example, in O. quadrata (419,
420), it reaches 116-212 mM and in Ocypode ceratopthalma
and Ocypode cordimanus under field conditions, it reaches
>40 mM and 27 mM, respectively. The primary urine of
O. quadrata is rather acidic (pH = 5.36±0.21), providing
an “acid trap” for NH4 + . On passage over the gills, the pH
is increased (pH = 7.01±0.24) and Cl− (but not Na+ ) is
reclaimed, such that the alkalinization of the fluid promotes
significant NH3 volatilization (∼71 μl. kg−1. h−1 ).
While pH 7 is not alkaline, the increase in pH is quite
effective, promoting a nearly 40-fold increase in potential
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Osmoregulation and Excretion
Comprehensive Physiology
Ocypode quadrata
Antennal
gland
Na+
H+
Urine
NH4+
NH4+
Na+ = 413 mmol . l–1
+
. –1
+
Na NH4 = 0.86 mmol l
pH
=
7.58
ATPase
NH3
Na+ = 255 mmol . l–1
NH4+ = 116 mmol . l–1
pH = 5.36
Hemolymph
Urine
Cuticle
Gill epithelium
NH3
Rh?
Increase of pH to 7.01
NH3
NH4+ + NH3
Cl–
HCO3–
NH3 (part. volatilized)
Metabolic
ammonia
NH3
Apical
Na+
+ ↔NH +/K+
NH3 + H
ATPase
4
NH4+/K+
+
H
H2O
HCO3– CO2
NH4+
CA
CA
Hemolymph
Figure 37 Ammonia excretion model in the terrestrial ghost crab Ocypode quadrata. The proposed mechanism is described in the text. The figure is modified after (1950).
diffusive gradient (Pnh3 ). In view of the parallel increase
in the fluid CO2 concentration and the uptake of Cl− , the
most obvious candidate for the net base excretion is transport
by an apical HCO3 − /Cl− exchanger (419). Reclamation of
urinary Na+ appears to be accomplished within the antennal
gland (420). These authors report high activity of Na+ /K+ ATPase in the antennal gland of O. quadrata for which NH4 +
may substitute for K+ in the basal membrane exchange. In
addition, apical Na+ /H+ antiporters in the antennal gland
may sustain both Na+ reclamation and acidification of urine
to promote NH4 + trapping (Fig. 37).
Studies of the more terrestrial grapsid crab, Geograpsus
grayi, have revealed modifications of the NH3 /NH4 + excretory system (646, 1904). G. grayi is a highly active carnivorous land crab and also reprocesses the urine to reclaim salts
via branchial uptake (646), but unlike Ocypode sp., it does
not employ ion reclamation within the antennal gland (1904).
Geograpsus grayi volatilizes NH3 from the limited volume
of “P” within the branchial chamber and thereby increases
the effective NH4 + capacity of the fluid, which may achieve
concentrations in excess of 80 mM compared with <1 mM
in the urine (1904). However, G. grayi manages a rate of
ammonia excretion comparable to that of aquatic crabs in
water (107-220 μmol. kg−1. h−1 [646, 1904]). Gaseous ammonia contributes ∼78% of this total excretion in a discontinuous process over 3 hours to 3 days (1904), although urine
flow is apparently limited, restricting fluid available for “P”
520
formation. The pH of this fluid (pH = 8.07) is higher than that
of the hemolymph (pH = 7.66-7.59), and at the same time the
CO2 content (36 mmol. l−1 ) is considerably greater than that
of the hemolymph (13.7-17.2 mmol. l−1 ). Amiloride reduced
NH4 + efflux by 83% in this system and reduced unidirectional
Na+ uptake. Thus, NH3 volatilization is achieved by raising
the fluid pH toward the pK such that gaseous NH3 becomes
8% of the total ammonia, creating a diffusive gradient (Pnh3
≈ 3.3 Pa) into the convective air stream (Fig. 38).
A net excretion reaction of
−
NH +
4 + HCO3 → H2 O + CO2 ↑ +NH 3 ↑
was proposed by Varley and Greenaway (1904). Since “acid
ammonia trapping” does not occur in the gills of this crab,
apical pathways for ammonia excretion might be similar to
marine or brackish-water crabs, possibly via exocytosis. In
fully terrestrial isopods, such as Porcellio scaber, NH3 is
volatilized from the surface of the abdomen, likely in a physiological coupling with active water vapor absorption (WVA)
(2037). Ventilatory movements of the pleopods are observed
during production of hyperosmotic fluid and WVA. Simultaneously with the production of WVA and pleopod movements, cyclic variations in total hemolymph ammonia were
observed (2036). It seems that elevated hemolymph ammonia concentrations occur only under advantageous conditions,
when atmospheric humidity allows WVA, and not humidity
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Comprehensive Physiology
Osmoregulation and Excretion
Geograpsus grayi
Urine
Cuticle
Gill epithelium
pH = 7.6
PCO2 = 0.93 kPa
NH3
Metabolic
ammonia
Rh?
pH = 8.07
PNH3 = 3.3 Pa
PCO2 = 1.87 kPa
NH4+
NH3
NH3 + H+ ↔ NH4+/K
Na+
NH3 ↑ + CO2 ↑ + H2O
NH4+/K+
H+/NH4+
Cl–
HCO3–
Na+
ATPase
NH4+
H+
H2O
HCO3– CO2
HCO3–
CA
CA
NH3 volatilised
Hemolymph
pH = ~7.6
Figure 38 Ammonia excretion model in the terrestrial crab Geograpsus grayi. The proposed mechanism
is described in the text. The figure is modified after (1950).
below of those appropriate for WVA. When conditions for
ammonia excretion are disadvantageous, ammonia is stored
as glutamine or arginine (2034, 2035). Transporters or mechanisms involved in hemolymph to surface ammonia excretion
are unknown so far. The fact that expression of an Rh-protein
was confirmed in the pleopods of the euryhaline isopod Idotea
baltica (Weihrauch unpubl. GenBank accession#: AY094181)
indicates that Rh-ammonia transporters most likely also play
a role in ammonia excretion in terrestrial isopods.
The single known exception for a crustacean that is not
ammoniotelic is the terrestrial anomuran robber crab Birgus
latro, which is purinotelic and excreting 79.5% of its total
excretory nitrogen as uric acid. The main route of excretion
is via the feces, accounting for nearly 96% of the total N
excretion (644, 645).
Urea excretion crustaceans With the exception of the
uricotelic land-living robber crab Birgus latro, crustaceans are
strictly ammoniotelic, and urea excretion usually accounts
for no more than 20% of the total nitrogenous waste (417,
449, 844, 969, 1302, 1944). Concentrations of urea in the
hemolymph are nonetheless relatively high and appear to be
salinity dependent (1944). As shown for the green shore crab
Carcinus maenas, hemolymph urea concentrations increase
with decreasing salinity from ∼20-80 μM in seawater to
∼600-1,000 μM in crabs inhabiting brackish water (10 ppt
salinity). In freshwater-living Chinese mitten crabs Eriocheir
sinensis, hemolymph urea concentration of ∼800 μM were
detected (1749, 1944). Moreover, an increase of hemolymph
urea in ammonia exposed juvenile E. sinensis suggests
urea synthesis, and therefore ammonia detoxification, as
an acute response to surging hemolymph ammonia during
high exposure (795). As mentioned above, an EST project
Volume 4, April 2014
employing cDNA generated from gills of the blue crab Callinectes sapidus revealed the presence of a putative branchial
urea transporter (Schaefer, EST project, GenBank accession
#: CV527852). Gill perfusion experiments on closely related
green shore crabs showed that urea is not excreted via the
branchial epithelia (1951). The physiological relevance of this
urea retention behavior in crabs acclimated to low salinities
remains puzzling, since maximal hemolymph urea concentration of around 1 mM in brackish-water-acclimated C. maenas
and freshwater-acclimated E. sinensis (1944) are likely too
low to be used for osmoregulatory purposes.
Insecta
As reviewed by Pant (1398), insects are considered in general to be uricotelic, as an adaptation to their rather terrestrial
lifestyle. In most terrestrial insects, 80% of the nitrogenous
waste is excreted as uric acid. Accordingly, the amount of
other nitrogenous waste products such as urea or ammonia
was thought to be small. However, as pointed out by Harrison and Phillips (703), in earlier days the amount of excreted
ammonia was likely strongly underestimated since the excretion products (pellets) were often oven-dried for analysis,
which led to volatilization and loss of gaseous ammonia. Further, procedures for the analysis of ammonia from excretory
pellets were not advanced enough to detect the molecule in
its precipitated form.
There are now a number of reports showing that excretion
of ammonia plays a considerable role as an excretion product in insects. Ammonotelism has been shown for aquatic
larval stages of insects, for example for lacewings, Sialis
lutaria (1750) and dragon fly nymphs of Aeshna cyanea
(1751). The freshwater-inhabiting larvae of the yellow fever
mosquito Aedes agypti also excrete ammonia. Employing
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Osmoregulation and Excretion
ion-selective electrodes, it was shown that ammonia excretion occurs in the osmoregulatory active anal papillae. It was
suggested that ammonia excretion occurs by ammonia trapping, since similar to the findings in freshwater fish gills (see
above), here also acidification of the unstirred boundary layer
was observed (442), mediated likely by the activity of the
apically localized H+ -ATPase (1407). It is conceivable that
hemolymph ammonia is actively transported into the epithelia
cell cytoplasm of the anal papillae via the basolaterally localized Na+ /K+ -ATPase (1407). It is noteworthy that two distinct Rh-like ammonia transporters were identified in Aedes
aegypti, sharing 87% identity in amino acid sequence to each
other and approximately 50% identity to trout Rhbg, in which
ammonia transport capability was verified. Both of these putative ammonia transporters are expressed in the anal papillae,
exhibiting a decrease in expression levels when larvae were
exposed to elevated environmental ammonia concentrations
(1948).
Excretion of ammonia by the larvae of terrestrial insects
has also been described. Larvae of the screwworm fly,
Wohlfahrtia vigil, infest open wounds in animal tissues and
were found to be ammoniotelic after feeding on flesh (210).
Ammonia excretion via the hindgut was shown for the larvae of the flesh fly Sacrophaga bullata, an insect that has
to deal with high loads of ammonia due to the ingestion
of rotting meat. The full mechanism of hindgut ammonia
excretion in this species is unknown, but excretion appears to
be active in the form of NH4 + (1490). Also for the tobacco
hornworm Manduca sexta (Lepidoptera), ammoniotelism was
suggested since high concentrations of ammonia (∼60 mM)
were found in the hindgut content (1943). In contrast to
the findings in Sacrophaga bullata, the hindgut of Manduca larvae does not exhibit active ammonia excretion. However, mRNA expression studies revealed high abundance of
an Rh-like ammonia transporter (RhMS). Since high mRNA
expression levels of RhMS and H+ -ATPase were found also
in the Mapighian tubules and tissues/lumen ammonia concentrations were high (∼25 mM; Weihrauch, unpublished data),
it was suggested that excess hemolymph ammonia is secreted
into the Mapighian tubules and then finally released for excretion into the hindgut (144, 1943). The species of the nitrogenous waste in insect larvae, however, depends on the food
source and availability of water. For instance, ryegrass fed
cranefly larva Tipula paludosa are clearly uricotelic, excreting close to four times more uric acid-N than ammonia-N
(650).
Adult terrestrial insects also excrete considerable amounts
of ammonia as summarized in detail by O’Donnell (1338)
and Weihrauch et al. (1948). For instance, in desert locusts,
ammonia concentrations in the feces were exceptional high
(up to 150 mM) (704), suggesting ammonotely. It was further shown that hindgut ammonia excretion was constant
over a luminal pH-range of 7-5 and sensitive to amiloride,
involving likely a Na+ /NH4 + transporter localized on the
mucosal side (1824). Although the presence of an Rh-protein
in the locust hindgut has not been shown yet, it is likely
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Comprehensive Physiology
that here also this ammonia transporter is abundant and plays
a role in ammonia excretion. Also cockroaches seem to be
ammoniotelic. As shown for the American cockroach Periplaneta americana, up to 90% of their nitrogenous waste is
excreted in the form of ammonia (1263). For blood-feeding
insects, the type of the main excretory product might differ. While the tsetse fly Glossina morsitans predominately
excretes uric acid and some amino acids such as arginine and
histidine (228), adult yellow fever mosquitos Aedes agypti
are ammoniotelic, excreting ammonia via the feces (1616).
In order to detoxify high ammonia loads derived from amino
acid metabolism after a blood meal, in addition to elevated
ammonia excretion rates, glutamine and proline are synthesized as temporary non-toxic nitrogenous storage molecules.
These amino acids are later utilized as important energy
sources (622).
Chelicerata
The primary nitrogenous waste product in spiders, scorpions and uropygids is guanine (31, 799, 1512). However, an
exception was found in Paruroctonus mesaensis. This desert
scorpion excretes more than 90% of its nitrogenous waste
the form of xanthine and is thereby xanthotelic (2058). The
pathway for nitrogenous waste in arachnids appears to be via
the Malpighian tubules/anal excretory system and not via the
urine produced by the coxal gland, which is predominantly
involved in ion and water balance (233, 234).
Urochordata
Many studies showed that ascidians, the most primitive living
chordates, are predominately ammoniotelic (626), excreting
up to 95% of their nitrogenous waste as ammonia. Beside this
excretion product, ascidians retain also substantial amounts
of uric acids and other purines, such as xanthine (627, 628,
1325). Some ascidians, but not Ciona intestinales or Styela
montereyensis, excrete considerable amounts of urea as well
(1158). At least in Styela plicata, urea excretion seems to
be temperature dependent, increasing with declining temperatures, thus leading to a shift from ammoniotelism to
ureotelism at temperatures below 20ºC (536). In other ascidians as well, however—for example, in Styela clava and Styela
partita—urea excretion is high, accounting for 47% and 42%
of total excreted nitrogen at 20ºC, respectively (1158). Ascidians are a particularly interesting species for analysis of nitrogen excretion since they bear gene information not only for
mammalian-like ammonia transporter Rhag, Rhbg, Rhcg, but
also for the rather primitive Rh-isoform RhP1 and at least two
distinct AMT ammonia transporters (809). While the function in ammonia excretion of all these potential ammonia
transporters needs to be evaluated, it was shown using gene
knockout techniques that Ci-AMT1a is essential for brain
development and brain function in Ciona intestinalis larvae
(1157).
Volume 4, April 2014
Comprehensive Physiology
Teleost fish
The subject of nitrogen excretion in fish has been reviewed and
investigated extensively in recent years by a number of groups
(72, 73, 191, 192, 813, 832, 1289, 1296-1300, 1608, 1873,
1927, 1928, 1953, 1996, 2041) and will only be summarized
here.
Freshwater teleosts The predominant pathway for
ammonia excretion in freshwater teleosts is by diffusion of
ammonia gas down its partial pressure (PNH3 ) gradient from
blood to water across the gill epithelium. A large driving
force in the current model of freshwater teleost ammonia
excretion is the V-ATPase-energized acidification of the gill
water boundary layer serving to readily protonate any NH3
arriving at this layer to form NH4 + , thus keeping the PNH3
in this boundary layer low (Fig. 34B). In addition, the hydration of CO2 , which crosses the gill membrane as CO2 gas
and forms H+ and HCO3 − , might also account for an apical acidification and ammonia trapping. For the rainbow trout
Oncorhynchus mykiss Nawata et al. (814) identified seven
full-length cDNAs, including one Rhag and two each of Rhbg,
Rhcg and Rh30-like. Rhbg and Rhcg-1 and -2 were expressed
in the gill. Functional expression analysis revealed that all
of the trout Rh-proteins, except Rh30-like, facilitated 14 Cmethylamine uptake when expressed in frog oocytes. Further,
by employing NH4 + -selective microelectrodes in conjunction with the scanning ion electrode technique (SIET), it was
confirmed for Rhag and Rhcg2 that these proteins indeed
mediate the transport of ammonia molecules. It was proposed
that in trout, Rh-glycoproteins are low-affinity, high-capacity
ammonia transporters (1300). Experiments exposing trout to
elevated environmental ammonia concentration gave the initial clues of the relevance of the different Rh-proteins and
other transporters in fish gill ammonia excretion. When fish
were exposed to 1.5 mM NH4 HCO3 in the surrounding water,
an initial uptake of ammonia into the plasma was observed,
resulting in increased plasma ammonia levels. When plasma
levels reached a certain threshold (∼500 μmol·l−1 ) after
approximately 12 hrs of exposure, ammonia net flux reverted,
showing a net excretion. After 48 hrs of exposure, however, plasma ammonia levels (∼1,000 μM) were only slightly
below the concentration in the water, and excretion rates were
highest. During this 48-hr period of ammonia exposure,
mRNA expression patterns of ammonia transporting proteins were quite different in branchial mitochondria-rich cells
(MRC) and pavement cells (PVC). While mRNA expression
of Rhbg, Rhcg1 and Rhcg2 did not change in MRC, a significant increase of mRNA expression of Rhbg (about 4-fold
after 48 hrs) and Rhcg2 (about 8-10-fold after 12 hr and 48
hr) was detected in PVC. Messenger RNA expression levels
of Rhcg1 did not change in PVC, but increased for the H+ ATPase (B-subunit) and Na+ /K+ -ATPase (α1a-subunit). In
contrast, mRNA for Na+ /K+ -ATPase in MRC revealed a significantly lower expression level after 48 hrs of exposure. In
Volume 4, April 2014
Osmoregulation and Excretion
both cell types, a decrease of mRNA coding for the cytoplasmic carbonic anhydrase-2 (CA2) was observed, concomitant
with decreased CA2 activity rates in whole cell homogenates.
This finding is puzzling since CA-catalyzed hydration of CO2
normally fueled the H+ -ATPase in trout gill (1059), which is
in turn important for branchial ammonia excretion (2007).
It was suggested that under exposure to high environmental ammonia, the H+ pump is fueled with protons generated
not by CA activity, but by dissociation of NH4 + that might
be transported at increased rates by the basolateral Na+ /K+ ATPase from the plasma into the epithelial cells. Direct evidence for participation of the Na+ /K+ -ATPase in ammonia
excretion in trout gill remains to be provided. However, it is
strongly suggested that the pavement cells play the dominant
role in branchial ammonia excretion. A hypothetical model
of branchial ammonia excretion in pavement cells is presented in Fig. 39. The presence of apically localized Na+ /H+
exchanger NHE2 and NHE3 in branchial Na+ /K+ -ATPasepositive MR cells in rainbow trout (835) led to the idea that
in freshwater fish, ammonia excretion and Na+ uptake function together in an apical Na+ /NH4 + exchange complex. It is
thought that this complex, which consists of a H+ -ATPase,
NHE2 and/or NHE3, Rhcg and Na+ channel, works together
as a “metabolon” that provides the acid-trapping mechanism
for apical ammonia excretion. Indirectly this metabolon is
supported by a carbonic anhydrase, providing H+ ions and
the basolateral Na+ /K+ -ATPase that accepts also NH4 + as a
substrate (2041). Such a metabolon can also be assumed for
the epidermal ammonia excretion mechanism in the planarian
S. mediterranea (1947, 2038, Fig. 35), and represents possibly a general mechanism for ammonia excretion in freshwater
animals.
Investigated by using a scanning ion-selective electrode
(SIET), it was shown that in freshwater zebrafish larvae (five
days post-fertilization), ammonia excretion occurs via the
skin, in this case specifically via H+ pump-rich cells and
to a minor extent in keratinocytes and other types of ionocytes in larval skin. Highest ammonia excretion rates were
detected in the yolk sac area. The NH4 + secretion was tightly
linked to acid secretion and was reduced when the low apical
pH generated by the proton pump was increased by a strong
buffer in the media or by direct inhibition of the H+ -ATPase.
A knockdown of Rhcg1 with morpholino-oligonucleotides
also reduced NH4 + secretion significantly, without effecting
H+ secretion (1665). All these findings indicate that ammonia
excretion in larval skin epithelia cells occur through an acidtrapping mechanism including the participation of Rhcg1.
A parallel study on zebrafish larvae measuring whole animal ammonia excretion after knockdown of Rhag, Rhbg or
Rhcg1 expression showed that silencing each of these Rhproteins caused, independently from each other, a reduction
of ∼50% in ammonia excretion rates confirming importance
of the Rh-proteins in the excretion mechanism in early life
history stages as well (191).
In addition to ammonia, zebrafish larvae also excrete urea
that at the life stages after hatching accounts for ∼20-30%
523
Osmoregulation and Excretion
Comprehensive Physiology
Oncorhynchus mykiss
Fresh water
Gill epithelium
Plasma
Gill boundary
water
CO2
CO2
CO2
Rhbg
NH3
HCO3–
+
H+
HCO3– + H+
CA
Rhcg2
NH3
NH3
H+-ATPase
NH4+
NH4+
NH4+
?
ATPase
Na+
Bulk water
–
NH3 + H+ –
NH4+
H+
Na+
Pavement cell
NH3
Basolateral
Apical
Figure 39
Branchial ammonia excretion model proposed for the rainbow trout
Oncorhynchus mykiss. The proposed mechanism is described in the text. The figure is modified after (1953).
of total N excretion (191). This urea excretion was reduced
by 90% relative to controls when the urea transporter (UT)
was silenced, underpinning the importance of UT in nitrogen
excretion. It seems that the synthesis of urea in freshwater
fish larvae serves as an important backup detoxification system for ammonia. Indeed, when ammonia excretion is compromised and reduced, for example, due to the knockdown
of Rhcg1, whole animal urea excretion rates increased nearly
twofold (191).
Also, for at least two adult teleost fish species—the
gulf toadfish (Opsanus beta) and the Lake Magadi Tilapia
(Alcolapia grahami)—it has been shown that some or all of
their nitrogenous waste is excreted as urea. The Lake Magadi
Tilapia excretes nearly all of its nitrogenous waste as urea
(1509) and exhibits a complete ornithine-urea cycle in several tissues (1068). In alkaline environments, the low levels
of protons in the external water reduce the conversion of NH3
to NH4 + , thus minimizing the outwardly dircted PNH3 and
thereby slowing the overall rate of NH3 excretion.
Marine teleosts Branchial ammonia excretion in marine
teleosts is considered to be quite different when compared
to freshwater fish. Ammonia excretion by ammonia trapping
due to the acidification of the unstirred gill boundary layer
is likely compromised in seawater because of the increased
buffering capacities of seawater. Also, since the gill of marine
fish is considered to be more permeable to small ions and
other molecules, paracellular pathways might play a role, in
addition to the substitution of NH4 + ions for K+ or H+ on
524
other ionic transporters. In the pufferfish Takifugu rubripes, a
typical seawater fish, Nakada et al. identified piscine homologues to Rhag, Rhbg, Rhcg1 and Rhcg2 (1289). It was
demonstrated that all isoforms are capable of the transport
of the ammonia analog, methylammonia, when expressed in
Xenopus oocytes. Interestingly, in T. rubripes, all Rh-proteins
were expressed in the gill, with Rhag expression in the gill
occurring in the pillar cells lining the vasculature and supporting the overlying pavement cells. Within the pavement
cells Rhbg was expressed in the basolateral membranes and
Rhcg2 was expressed in the apical membranes. Further, while
Rhcg1 was expressed in the apical membrane of the chloride
cells, no Rh proteins could be detected in the basolateral
membranes of this cell type. Given the very large fractional
surface area of the gill that is covered by pavement cells,
one can predict that these cells will dominate in both mode
and quantity of ammonia transported. Experiments exposing
pufferfish T. rubripes to high environmental ammonia (1 mM
NH4 HCO3 ) provided further insights into the suite of transporters likely involved in branchial ammonia transport. Under
these conditions plasma ammonia concentrations increased
significantly from ∼350 μM to ∼800 μM. At the same time
mRNA expression of branchial Rhbg was downregulated,
while MRC Rhcg1 was strongly upregulated after 48 hrs of
exposure. It should be mentioned that under these conditions
ammonia was still excreted at rates similar to control rates.
Along with changes in Rh gene expression, there was also
an upregulation of the branchial H+ -ATPase, NHE3, NKCC
and the Na+ /K+ -ATPase (1296). Both Na+ /K+ -ATPase and
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Osmoregulation and Excretion
NKCC have been shown to be expressed in MRC in seawater fish gill (885, 1201, 1417, 1905). No evidence for gill
cell specific localization of H+ -ATPase and NHE3 has been
presented to date for seawater fish; however, apical localization of these transporters in MRC of freshwater fish suggest
a similar cell specificity in seawater fish, at least when the
fish are ammonia stressed. According to Nawata and others
(1296), ammonia stress activates ammonia excretion mechanisms in MCR, where ammonia is transported actively via the
Na+ /K+ -ATPase from the plasma into the MRC cytoplasm.
Direct participation of the Na+ /K+ -ATPase in ammonia transport was supported by enzyme assays that showed that this
pump exhibited high activity when K+ ions were replaced
with NH4 + . Plasma-to-cytoplasm transport of ammonia via
basolateral NKCC might be driven by the Na+ /K+ -ATPase
at the same time. The upregulation of H+ -ATPase and NHE3
(NHE2 is downregulated under ammonia stress) suggests an
ammonia-trapping mechanism on the apical side by acidification of the unstirred gill boundary layer. This speculation
is supported by the fact that in seawater fish, MRC is excreting ions into an apical crypt (514), a morphological structure
composed of MRC and accessory cells, where water exchange
by ventilation is likely limited and seawater pH buffer capacities are easily exhausted. Presence of an apical localized
H+ -ATPase is at this point speculative and needs to be verified (Fig. 40). An apical localization of NHE3 in MRC of
the amphibious mudskipper Periophthalmodon schlosseri is
known (508), and at least under environmental ammonia stress
conditions, it was suggested for this fish that active ammonia
is mediated via an apical NHE and Na+ /K+ -ATPase (1508).
Also in this species an apically localized H+ -ATPase was
detected in MRC (2006). Further, the evidence of expression
of Rhbg and Rhcg2 in the skin of T. rubripes suggests that,
in addition to the gills, the skin also plays a certain role in
ammonia excretion in marine fish (1296).
In addition to ammonia, substantial excretion of urea has
been found in the gulf toadfish Opsanus beta, a saltwater
teleost. Opsanus beta can excrete urea in distinct pulses
mainly through a UT-mediated pathway in the gills (1204,
2021), and in addition to Rhbg, Rhcg expression of an functional urea transporter (tUT) has been verified in the gills of
the fish (1885) (1906). In their natural habitat gulf toadfish
excrete roughly similar amounts of ammonia and urea (72,
73). Some evidence suggests that urea excretion functions
to hide the scent of ammonia to predators by the cloaking
molecule urea (73).
Ammonia excretion in amphibious teleosts In air,
NH3 cannot be easily hydrated, which would negate ammonia trapping across epithelial tissues such as the skin or gills.
Amphibious fishes deal with these problems by either tolerating extremely high internal concentrations of ammonia,
found in fishes such as the oriental weatherloach (Misgurnus
anguillicaudatus; [1874]), or by detoxifying ammonia to less
toxic waste end-products such as glutamine or urea as is seen
in the lungfishes (282, 842, 843, 1715). There are a number of
fishes able to excrete ammonia in air. The giant mudskipper
(Periopthalmodon schlosseri) and the climbing perch (Anabas
testudineus) appear to use active NH4 + excretion to excrete
Takifugu rubripes
Gill epithelium
Plasma
Rhag Rhbg
Rhag
Sea water
Rhbg2
NH3
Pillar
cell
NH4+
Na+
2Cl–
NH4+/K+
NH4+
PV cell model
Na+
ATPase
MR cell model
Na+
NH4+
NH3 + H+
Rhbg1
NH3
Na+
K+
Basolateral
ATPase
NHE-3
H+
H+-ATPase ?
NH4+
H+
Apical
Figure 40
Ammonia excretion model proposed for pavement and mitochondria-rich cells
in the puffer fish Takifugu rubripes. The proposed mechanism is described in the text. The
figure is modified after (1953) and (1289).
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Comprehensive Physiology
ammonia against massive inwardly directed PNH3 and NH4 +
electrochemical gradients (283, 1508, 1809).
The giant mudskipper is an obligatory air-breathing fish
that drowns if denied access to air (1508). Experiments
employing ouabain suggested basolateral NH4 + transport
takes place via the Na+ /K+ -ATPase (Randall, 1994), which
possibly also powers basolateral NH4 + uptake by a NKCC
cotransporter (950, 1506). Both transporters have been localized to the basolateral membrane of MRC via immunohistochemical studies (2006). Ammonia excretion is also amiloride
sensitive, suggesting an apical Na+ / H+ (NH4 + ) exchange via
NHE. The retention of saltwater within the confines of chambers formed by the fused lamellae of in the gill of the giant
mudskipper possibly provides the needed inwardly directed
Na+ electrochemical gradient to energize the process (2003).
Localization of an H+ ATPase to the apical crypt region of
MRC suggests that this pump also participates in ammonia
excretion, probably by ammonia trapping. However, buffering
the apical solution had no effect on the excretion rates (2006).
In contrast, partial reduction of the ammonia excretion rate
was accomplished employing acetazolamide, indicating the
participation of the carbonic anhydrase in this process.
Although not demonstrated yet, it is likely that in the
MRC of the mudskipper, as shown for MRC in other teleost
fish, an apically Rhcg1 is present and plays a role in ammonia excretion. Experiments by Chew and others showed that
only a small percentage of plasma ammonia is detoxified to
urea during emersion (283) (Fig. 41). Ammonia volatilization
across the skin of amphibious fish has been reported for the airbreathing tropical fishes, the Mangrove killifish Kryptolebias
marmoratus (557, 558) and the oriental weatherloach Misgurnus anguillicaudatus (1874). The weatherloach prefers
the muddy or sandy bottoms of lakes, ponds and rice fields,
and will migrate overland when necessary if water is scarce
(833). Tsui et al. (1874) showed that the skin of M. anguillicaudatus becomes alkalinized by approximately 1.6 pH units,
leading to the suggestion that this fish volatilizes ammonia
as NH3 during air exposure. Although traces of volatilized
NH3 were measured, the overall importance of cutaneous
ammonia excretion in this animal is not clear. To achieve
ammonia volatilization, plasma ammonia levels would have
to reach very high concentrations to generate the needed PNH3
gradients. In fact, the oriental weatherloach is able to tolerate plasma total ammonia concentrations of ca. 5 mmol·l−1 ,
making it one of the most ammonia-tolerant fishes known so
far (281, 1874).
The mangrove killifish K. marmoratusi inhabits intertidal zones and mangrove swamps, and may occasionally be
immersed as it hides among leaves and other debris in this
habitat (557). K. marmoratusi volatilizes NH3 across its body
surface when exposed to air under humid conditions, likely to
prevent or minimize the accumulation of ammonia within the
tissues (557, 1081). It was shown by (558) that approximately
40-50% of the ammonia excreted by this animal during air
exposure was across the back end of the body. During air exposure the cutaneous surface is alkalinized by 0.4 to 0.5 units
(1081), thus promoting the accumulation of NH3 on the skin
surface. It was proposed that slight air currents would be sufficient to reduce the boundary layers and volatize the NH3 at
the skin surface, where a PNH3 of approximating 1200 μTorr
was measured.
The mode of ammonia excretion across the skin of the
mudskipper is still puzzling. Hung et al. (814) demonstrated
mRNA presence of Rhbg, Rhcg1 and Rhcg2 in the skin of the
fish, of which Rhcg1 and Rhcg2 were upregulated several-fold
when fish were exposed to air (24 hrs). Assuming that Rhcg1
Periophthalmodon schlosseri
Gill epithelium
Plasma
NH4+
NH4+/K+
Na+
ATPase
Na+
2Cl–
+ +
NH4 /K
NH3
Sea water
Na+
H+/NH4+
Rhcg1?
NH3
NH3
H+
CO2
CA
HCO3
MR cell
Basolateral
NHE
NH4+
H+
–
Cl–
Apical
Figure
41 Branchial ammonia excretion model proposed for
mitochondria-rich cells in the giant mudskipper Periophthalmodon
schlosseri. The proposed mechanism is described in the text. The figure
is modified after (1953).
526
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Osmoregulation and Excretion
Kryptolebias marmoratus
Plasma
Epithelium
(pHe = 7.7)
Cutaneous
surface
(pHi = 7.1 to 7.4)
CO2
(pHskin = 7.49–7.84)
CO2
CO2
Cl–
CA?
Na+
H+ +
–
HCO3
NHE
Rhcg1
Rhbg
NH3
NH4+
Air
NH3
NH4+
Na+
2Cl–
NH4+/K+
Rhcg2
NH3
Na+
ATPase
?
NH4+
Basolateral
NH4+
Apical
Figure 42
Proposed ammonia excretion across the skin of the Mangrove killifish Kryptolebias marmoratus. The proposed mechanism is described in the text. The figure is
modified after (1953).
and Rhcg2 promote NH3 transport, apical alkalinization of
the skin could be accomplished either by apical NH3 excretion or HCO3 − secretion (the latter mediated by the action
of an intracellular carbonic anhydrase) or both mechanisms.
It is likely that for this mechanism to function, intracellular
H+ generated by CA must be eliminated back into the plasma
possibly via a basolateral NHE. It was speculated that basolateral plasma to cytoplasm ammonia uptake occurs actively
via the Na+ /K+ -ATPase and maybe NK(NH4 + )CC cotransport, which could provide an apical outwardly directed ammonia gradient (1081). Basolateral participation of a Na+ /K+ ATPase in ammonia excretion is indeed conceivable since
most animal epithelia cells express this pump, which is apparently involved in most active ammonia transport mechanisms,
as reviewed in this article. A hypothetical model of cutanous
ammonia excretion in the mangrove killifish is illustrated in
Fig. 42.
Elasmobranchs As elaborated in a separate section of
this article, marine elasmobranch fishes, such as sharks, rays
and skates, retain more than 300 mM urea in the extracellular
fluids, which is used as osmotic ballast (1716). As a consequence, a large concentration gradient for urea exists between
the plasma fluids and the environment, resulting in a passive
loss of urea (164). It is because of this leakage that marine
elasmobranchs are ureotelic. For instance, in the spiny dogfish
Squalus acanthias, more than 90% of the nitrogenous waste
is excreted in the form of urea and less than 5% as ammonia
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(2021). The main site of urea excretion is the gills, accounting for nearly 95% of the total urea loss from the fish (2021).
Although urea transporters have been identified on a molecular basis in elasmobranchs (34), expression in the gills has not
been demonstrated. In contrast, experiments on basolateral
membrane vesicles from dogfish gills suggest the presence a
phloretin-sensitive sodium-coupled urea transporter, thought
to be involved in the reclamation of urea that has leaked into
the epithelia cells (531).
The role of ammonia excretion in elasmobranchs is not
well understood yet. For pups of the spiny dogfish it was
calculated that about 77% of total branchial ammonia efflux
was via non-ionic diffusion of NH3 and ∼6% as diffusion
of NH4 + , likely paracelluar (511). Employing bumetanide,
a small but significant inhibition of ammonia excretion was
detected, suggesting the participation of a basolateral NKCC.
In the gills of the chondrichthyan ratfish Hydrolagus colliei,
molecular evidence for an Rh-like ammonia transporter was
found (36). Whether this branchial ammonia transporter is
used for ammonia excretion or ammonia retention remains to
be investigated.
Amphibia The subject of nitrogen excretion in amphibians has been studied by many investigators, with the most
recent review by Wells (1964). Amphibians exhibit an exceptional plasticity in their nitrogen excretion strategies. For
instance, the Bornean toad Bufo quadriporcatus dwells mostly
on the forest floor and is ureotelic when on land, but switches
to ammonotely when placed in water (1674, 1964). Other
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Osmoregulation and Excretion
amphibians, for example the toad Bufo asper or the frog
Limnonectes blythii, show a lesser degree of flexibility with
regard to the form of nitrogen excreted. Both species are
usually found along river banks; however, they also remain
ureotelic when placed in water (1674). Embryos and larvae
of anurans and urodeles are usually aquatic and commonly
excrete ammonia as the primary nitrogenous waste product.
However, larvae of species that develop in dry conditions or
in surroundings with only limited water exchange are found
to be ureotelic. For instance, larvae of the Victorian smooth
froglet Geocrina victoriana, a species with terrestrial eggs,
excrete up to 86% of their nitrogenous waste as urea (1171).
Also, embryos of amphibians developing in pouches on the
back of a marsupial frog such as Gastrotheca riobambae (17)
or in the uterine fluid inside of the reproductive tract of the
ovoviviparous Salamandra salamandra (1621) are ureotelic.
Some frogs, for example the Argentinian frog Leptodactylus
bufonius (1675) or the African pig-nosed frog Hemisus marmoratus, lay their eggs in foam nests, where their larvae
develop. The tadpoles of H. marmoratus excrete about 80%
of the nitrogenous waste in form of urea (637). Clearly exceptional for amphibians is the excretion of uric acid as the main
nitrogenous waste product. Uricotely has been decribed so far
only in “waterproof frogs” in the genera Chiromantis (e.g.,
the South African Grey foam-nested tree frog Chiromantis
xerampelina) and Phyllomendusa (1100, 1668, 1672). The
arboreal Waxy Monkey Leaf Frog, Phyllomedusa sauvagii,
lays its eggs not into water bodies but on leaves. During early
stages of development, tadpoles of P. sauvagii excrete similar
amounts of ammonia and urea, but gradually switch toward
ureotely in later stages. While ammonia excretion rates further decrease over time, uric acid release is initiated and can
be detected even before completion of metamorphosis (1673,
1964). Semiaquatic and terrestrial amphibians are generally
considered to be ureotelic when reaching adulthood. However,
along with urea, some ammonia is excreted (351, 2031). Fully
aquatic frogs, such as Xenopus laevis, or amphibians that do
not complete metamorphosis from fully aquatic larvae stages
to the “adult” semiterrestrial life form, such as the mudpuppy
Necturus maculosus, remain ammoniotelic (351, 516, 2031).
In typical frogs such as Rana, ammonia excretion occurs
in the kidney by an active secretion in the distal tubules and
collecting ducts (1920). However, in fully aquatic and semiaquatic amphibians, ammonia is also partially excreted across
the skin (68, 551, 582, 1766, 1903). In the mudpuppy Necturus maculosus, up to 90% of body fluid ammonia is excreted
via the skin, and 10% ammonia plus urea is eliminated by the
kidney (516). The aquatic caecilian “rubber eel” Typhonectes
natans excrete similar amounts of their nitrogenous waste in
form of ammonia and urea, with both molecules (∼90% of
ammonia and ∼70% of urea) being predominately eliminated
via the skin (1766). Also for semiaquatic frogs such as Leptodactylus ocellatus (1573), Rana esculenta (1493) and the
Northern Leopard frog Rana pipiens, ammonia excretion via
the skin was documented. Skin excretion rates in R. pipiens
could be stimulated by applying an internal ammonia load
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or by plasma obtained from a dog during metabolic acidosis (551). The precise mechanism responsible for ammonia
excretion via the amphibian skin is unknown at this moment.
However, it is conceivable that ammonia is eliminated by
ammonia trapping in a similar fashion as described in freshwater fish gills (see above). The model illustrating the hypothesis for osmoregulatory NaCl uptake across frog skin (see section on Amphibia, Fig. 21) depicts two cell types: MR cells
containing an apical H+ -ATPase and basolateral Na+ /K+ ATPase, and electrically coupled principal (granular) cells
containing a basolateral Na+ /K+ -ATPase. Moreover, genome
projects and ESTs on Xenopus tropicalis and Xenopus laevis
revealed the presence of genes coding for the ammonia transporters Rhag, Rhbg, Rhcg and RhP1 (809). Indeed, a recent
study revealed that cutaneous ammonia excretion in X. laevis (∼50% of total excretion) is active and can be enhanced
by the phosphodiesterase blocker theophylline. Gene expression analysis and enzyme activity assays strongly suggest the
participation of the Na+ /K+ -ATPase, H+ -ATPase, Rhcg and
Rhbg in the cutaneous excretion process (362).
Experiments in the neotenic axolotl Siredon mexicanum
suggest further that the gills, especially in smaller animals
with well-developed vascular gills, are able to excrete waste
nitrogen (1264).
Conclusions
The energetics of ion and water exchanges in aquatic animals constitutes one of the main themes of the article. Several
types of ion motive ATPases are expressed in osmoregulatory organs of invertebrates and vertebrates. Among these
ATPases, the distribution and function of the Na+ /K+ -ATPase
discovered in the late 1950s have been studied in all major
organs specialized for whole body osmoregulation such as
kidneys, gills, antennal glands, integument, intestine and
exocrine glands. In all animals studied, the “sodium pump”
energizes the high extracellular Na+ /K+ and low intracellular Na+ /K+ concentration ratios. Cell water volume is
determined to a large extent by the intracellular Cl− pool
maintained above thermodynamic equilibrium by the concerted and regulated activity of several mechanisms such as
the Na+ /K+ pump, the Na+ -2Cl− -K+ cotransporter and the
passive flows of Cl− and K+ in specific channels in the
plasma membrane. Studies within the last decades have indicated that the V-type H+ pump, which plays a central role in
energizing insect epithelial ion transport, plays specific roles
in extracellular osmoregulation and acid-base balance of animals generally. In all epithelia investigated, the Na+ /K+ pump
is expressed at the membranes lining the lateral intercellular
spaces that would allow for solute-coupled water transport,
isotonic transport and uphill water transport. These functions
have been studied in proximal nephron of vertebrate kidney, small intestine and anuran skin, which have been shown
to transport water at transepithelial osmotic equilibrium.
Experimental studies and quantitative biophysical analyses
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of cellular energy expenditure in freshwater invertebrates and
amphibians indicate that the concerted activity of the two
above-mentioned ion motive ATPases would allow for uptake
of NaCl from concentrations as low as 10−5 mol·l−1 . In some
epithelia, additional ion motive ATPases fuel specific epithelial functions, as, for example, the Na+ /NH4 + ATPase in crustacean gills.
The comparative approach applied throughout this article permits the identification of common principles and considerations of the diversity of the cellular organization of
osmoregulatory and excretory epithelia. This identification of
common principles within diverse organization constitutes a
second main theme of the article. The polarized epithelia constituting the interface between the extracellular fluid and the
environment are multifunctional by serving extracellular ionand water homeostasis, acid-base balance, and excretion of
nitrogenous waste products. The associated thermodynamic
work is fueled by hydrolysis of ATP at Na+ /K+ pumps and
H+ pumps. Different aquaporin water channels are expressed
at the luminal and basolateral membranes, and the regulation
of transepithelial water transport takes place by trafficking of
aquaporins between cytoplasmic pools and the apical membrane. Across taxonomic groups, osmoregulatory epithelia are
multicellular with minority cell types, denoted intercalatedor mitochondria-rich (mitochondrion-rich) (MR) cells, interspersed between principal cells. Several types of MR cells
have evolved not only across different phyla but also within a
given taxonomic group as exemplified in the gills of freshwater teleosts. Special attention is paid to hypoosmoregulators in
marine environments that depend on salt-secreting exocrine
glands (reptiles, birds), gills (crustacea and teleost fish) and
newly recognized specialized intestinal epithelia (teleost fish).
Beyond the well-described segmental functional organization
of the vertebrate nephron there are other examples of tightly
coupled coordinated ion and water transport across epithelia
associated anatomically, such as the complex of Malpighian
tubule, midgut and rectum of insects, and probably also the
epidermal epithelium and the subepidermal exocrine glands
in the skin of frogs on land.
The third major theme of the article concerns whole body
ion and water homeostasis resulting from balanced uptake
and elimination of water and solutes governed by different
osmoregulatory organs. The discussion aims at the diversity of adaptations and the mechanisms of acclimatization in
species living in and migrating between environments of different osmotic conditions. The conclusions are summarized
for individual taxonomic groups as outlined below.
Annelida and Mollusca
Most molluscs that live in seawater are osmotic conformers; these animals conserve energy at the organismal level by
allowing the ionic concentration of the extracellular fluid to
vary with the ambient concentration. The cost of this strategy
is that each cell in the animal must retain the mechanisms
and capacity to either increase or decrease the cytoplasmic
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Osmoregulation and Excretion
osmotic concentration. Cellular regulation involves changes
in cytoplasmic concentrations of both inorganic ions and
organic molecules, such as amino acids. Species that live
in brackish water often are osmotic conformers at higher
salinities and hyperosmotic regulators at ambient salinities
below 4-5. At lower salinities, the osmotic and ionic gradients between ambient and the body fluids are low compared
to that in arthropods and the energetic cost of ion uptake
and production of the hypo-osmotic cytoplasm is reduced.
Terrestrial molluscs and annelids are subject to desiccation,
and can survive the loss of considerable amounts of body
water by evaporation. Withdrawal into the shell (if present)
reduces water loss. These animals can take up water by osmosis across the body wall. Urine is produced by filtration. In
annelids, nephridia are present and the number in each animal
varies greatly among species across the phylum. In molluscs,
the heart is the site of filtration and the filtrate is processed by
a kidney.
Crustacea
The vast majority of crustaceans are aquatic animals of marine
origin. Most marine crustaceans are osmoconformers, maintaining only minor osmotic gradients across their body surface. However, a number of these osmoconformers is considerably euryhaline, having the ability to migrate to more
dilute ambient media on the basis of their capability for intracellular osmoregulation, adjusting the intracellular osmotic
concentration to the values of the hemolymph and the ambient medium. Hyperosmoregulating crustacians evolved mechanisms for extracellular osmoregulation, allowing them to
inhabit more dilute ambient media like freshwater and more
diluted brackish waters. Apart of preventive mechanisms like
reduced body surface permeabilities and the tolerance of
reduced body fluid osmolalities, hyperosmoregulating crustaceans evolved transport mechanisms in branchial epithelia that compensate for the passive loss of salt to the dilute
ambient media. Two different mechanisms of active NaCl
absorption have been characterized. Like amphibians and fish,
freshwater crustaceans appear to use epithelial Na+ channels
and V-type H+ -ATPases in the apical membrane of the gill
epithelium. On the other hand, a mechanism involving apical
Na+ -2Cl− -K+ cotransporters and K+ channels and/or electroneutral cation and anion exchangers appears to dominate
active NaCl absorption in hyperosmoregulating crustaceans
in moderately dilute ambient media. The passive water gain
in dilute waters is balanced by urine production. Whereas
the latter considerably contributes to the overall salt loss in
many hyperregulating crustaceans without capability to produce dilute urine, some crustaceans evolved transport mechanisms in the antennal glands to reduce this loss of salt by
active absorption. Some crustaceans are hypoosmoregulators
in the sea or in hypersaline media. It appears that the gills
are the site of active salt secretion. However, the mechanisms
are unknown and require further study. Whereas the gills are
the major site of osmotic and ionic regulation in Crustacea,
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Osmoregulation and Excretion
the antennal glands, the gut and the hepatopancreas contribute
to osmotic, ionic and pH homeostasis.
Insecta
The increased availability of oxygen and novel food sources
in the form of flowering plants were important factors contributing to the explosive population expansion and speciation
of terrestrial insects. Terrestriality also created the potential
for desiccation, however, particularly for small animals with
a high ratio of surface area to volume. There was thus a
strong selection pressure for the evolution of extraordinarily
effective mechanisms to reduce water loss from the gut, from
the respiratory system and across the cuticle of the exoskeleton. Lipids in the epicuticular (outer) layer of the cuticle are
the primary barrier to transpiratory water loss, analogous to
the role of epidermal lipids in limiting water loss by reptiles (see Reptiles, below) and some terrestrial amphibians
(see Amphibians, below). Along with the evolution of flight,
these mechanisms for restricting water loss were major factors contributing to the success of insects on land. In contrast to the dominant role of the Na+ /K+ -ATPase in powering ion transport by vertebrate osmoregulatory epithelia, the
vacuolar-type H+ -ATPase plays a preeminent role in driving
secretion or absorption of ions across the plasma membranes
of the gut and Malpighian (renal) tubules of insects. It is
also worth noting that although plants provided potential food
sources for insects, flowering plants evolved numerous toxins
designed to minimize herbivory. In turn, powerful mechanisms for detoxification and excretion of such compounds
have evolved in the insects. Microarray studies show that the
genes for organic solute transporters dominate the array listing for the Malpighian tubules, consistent with a pivotal role
for the tubules in elimination of toxins.
Agnata and Pisces
While freshwater fish are forced to regulate internal salt concentrations above ambient, all osmoregulatory strategies are
represented among marine fishes. A high number of teleost
species are euryhaline and osmoregulate efficiently in salinities ranging from freshwater to seawater, offering convenient models for the study of osmoregulatory physiology.
Among these species killifish, several tilapia and salmonids
have become model organisms. Much of what is known about
osmoregulation by teleosts stems from this limited number
of species as well as zebrafish, commonly used for studies of
freshwater osmoregulation. Even within this limited number
of species, it is already apparent that distinct mechanisms for
ion uptake in hypoosmotic environments exist. Undoubtedly
this diversity reflects a high number of freshwater invasions,
and it follows that exploration of additional species in comparative approaches will yield additional insight into mechanisms
responsible for freshwater osmoregulation in the most radiant group of vertebrates. The past few years have revealed
novel insight into the mode by which ammonia excretion
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Comprehensive Physiology
and Na+ uptake is linked in freshwater fish. It is becoming
clear, however, that this aspect of brachial transport in freshwater fish varies among species as well. So far it appears
that mechanisms of salt and water transport are more consistent among marine species. However, this may simply reflect
that significantly less attention has been devoted to marine
species. Recent years have witnessed a departure from the
tradition of doing studies of fish osmoregulation on unfed
animals and have already revealed important roles of dietary
salt intake in freshwater species as well as complicated interactions between processing of ingested food and osmoregulation in marine species. Epithelial barrier function afforded
by claudins and occludin is arguably one of the most exciting
novel areas of fish osmoregulation and is bound to receive
considerable attention over the next decades.
Amphibia
Modern amphibians are adapted to almost all climatic zones
and are found in the range from purely aquatic to purely terrestrial habitats. Generally, the body fluids are diluted as compared to other vertebrates, with ion concentrations submitted
to larger variations. In freshwater, a high rate of glomerular filtration secures the cutaneous uptake of water being balanced
by voiding of diluted urine. The water permeability of the urinary bladder is small, and reabsorption of water from the bladder urine is of marginal importance for body water balance.
In dry environments, urine production decreases because of
decreased glomerular filtration rate and increased tubular fractional water reabsorption. Urine is stored in the bladder and
recycled into the body fluids governed by evaporation from
the body surface. In anurans, water is taken up by cutaneous
drinking, which at all levels, from behavior to the anatomical
and functional organization of the skin, is an advanced specialization of terrestrial living. The regulation of water and ion
exchange at the cell level of skin, kidney and urinary bladder
has been studied extensively, leaving unanswered questions
about how their functions are coordinated at body level. Adaptations at organ and cell levels have evolved specifically for
living alternately in terrestrial and aquatic habitats. In freshwater, uptake of Na+ by the principal cells and of Cl− by
MR cells is both active and fueled by the P-type Na+ /K+ ATPase and the V-type H+ -ATPase, respectively. The frog on
land maintains a slightly hypotonic cutaneous surface fluid by
regulated subepidermal gland secretion, which is balanced by
water evaporation into the atmosphere and ion and water reabsorption by principal and MR cells of the epidermis. These
adaptations evolved pari passu with dynamic permeabilities
of the skin controlled by external ion and osmotic concentrations. This allowed for fast switching of the cutaneous uptake
of chloride between active and passive transport associated
with a dynamic electrical coupling of active sodium uptake
by principal cells and passive chloride uptake by MR cells.
It is not known whether similar adaptations are present in all
anurans and in salamanders and caecilians.
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Comprehensive Physiology
Reptilia
Although the solute and water composition of the milieu
intérieur is not as tightly regulated in reptiles as in mammals and birds, it is still maintained within certain limits.
The skin, kidneys, bladder, cloaca, colon and extrarenal salt
glands all play important roles in this process. The function of
these organs and their regulation with changes in the external
environment differ among orders and species from different
habitats. Much is known about the function of these organs,
but there are still major gaps in our knowledge. Moreover,
although significant information is available about the regulation of many specific routes of solute and water movement,
the information is far from complete, and almost nothing is
known about the comprehensive integrated regulation of all
these routes together. Such integrated regulation is essential
to the overall maintenance of the milieu intérieur.
Aves
Birds have sufficient osmoregulatory ability to inhabit all
environments on earth while maintaining their water and electrolyte milieu intérieur within very narrow limits. Osmoregulation involves both renal and extrarenal mechanisms. Renal
mechanisms involve changes in GFR, including changes in
the number of filtering nephrons; changes in tubular reabsorption of salt and water, including production of a moderately
concentrated urine; and excretion of uric acid as the primary
end product of nitrogen metabolism. Extrarenal mechanisms
involve modification of ureteral urine in the lower gastrointestinal tract and secretion of a concentrated sodium chloride
solution by nasal salt glands. Although urine concentrating
ability is limited, excretion of uric acid and the solute-coupled
water absorption in the lower intestine make this limited concentrating ability highly appropriate to the conservation of
water, and the nasal salt glands permit the additional excretion of sodium chloride and production of solute-free water
in those marine species that require it. However, much still
remains to be learned about the integration of these osmoregulatory mechanisms in the intact animal.
Osmoregulation and Excretion
amphibians. This section reveals that our knowledge on nitrogen excretion and its underlying mechanisms in aquatic and
terrestrial animals is by far not complete. Among other things,
future studies will show whether or not H+ -ATPase and Rhprotein-mediated ammonia trapping is a general excretion
strategy in freshwater animals and further reveal the relevance of a vesicular ammonia transport mechanism in animals inhabiting environments with a higher buffer capacity.
Open questions also remain regarding the role and function
of AMTs and aquaporins in nitrogen transporting epithelia.
Author Contributions and Acknowledgments
Erik Hviid Larsen wrote Introduction, Biophysical Concepts
in Osmoregulation and Amphibia (supported by Carlsberg
Foundation 2008-01-0639). Lewis E. Deaton wrote Annelida and Mollusca (comparative research was supported by
National Science Foundation). Horst Onken wrote Crustacea
(comparative research was supported by the National Institutes of Health and by Wagner College Research Grants).
Michael O’Donnell wrote Insecta (supported by the Natural
Sciences and Engineering Research Council of Canada). Martin Grosell wrote Agnata and Pisces (supported by National
Science Foundation, IOB 1146695). William H. Dantzler
wrote Reptilia and Aves (comparative research for both was
supported by National Science Foundation). Dirk Weihrauch
wrote Nitrogen Excretion (supported by Natural Sciences and
Engineering Research Council of Canada). All authors wrote
the Conclusions section and approved the manuscript.
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